Barley with low levels of hordeins

ABSTRACT

The present invention relates to methods of producing a food or malt-base beverage suitable for consumption by a subject with Coeliac&#39;s disease. In particular, the present invention relates to methods of producing a food or malt-based beverage with low levels of hordeins. Also provided are barley plants which produce grain that can be used in the methods of the invention.

This application is a continuation of U.S. Ser. No. 12/733,139, filedMay 10, 2010, now allowed, which is a §371 national stage of PCTInternational Application No. PCT/AU2008/001172, filed Aug. 13, 2008,which claims priority of U.S. Provisional Application No. 60/964,672,filed Aug. 13, 2007, the content of each of which is hereby incorporatedby reference in its entirety into this application.

FIELD OF THE INVENTION

The present invention relates to methods of producing a food ormalt-based beverage suitable for consumption by a subject with Coeliac'sdisease. In particular, the present invention relates to methods ofproducing a food or malt-based beverage with low levels of hordeins.Also provided are barley plants which produce grain that can be used inthe methods of the invention.

BACKGROUND OF THE INVENTION

Coeliac (celiac) disease (CD, also called celiac sprue) is a T-cellmediated autoimmune disease of the small intestine which is triggered insusceptible individuals by ingestion of particular storage proteins,collectively known as prolamins, from wheat (gluten consisting ofglutenins and gliadins), barley (hordeins) or rye (secalins). Oatprolamins (avenins) appear to be tolerated by the majority of coeliacs(Hogberg et al., 2004; Peraaho et al., 2004a) but may induce positivereactions in a minority of coeliacs (Lundin et al. 2003; Peraaho et al.2004b). CD occurs in approximately 0.25-1% of the population in at leastAustralia, North and South America, Europe, Africa and India (Hovell etal. 2001; Fasano et al. 2003; Treem 2004) but the disease is probablyunderdiagnosed. Increased awareness of the symptoms and consequences ofuntreated CD has lead to rates of diagnosis in Australia increasing at15% per year. About 1 in 4 Caucasians and West Asians carry the HLA-DQ8or -DQ2 alleles which are a necessary but not sufficient determinant ofCD (Treem 2004). However, only about 1 in 20 people with these allelesdevelop CD. At present the only treatment is total avoidance of wheat,barley and rye, as recurrences may be triggered by consumption of aslittle as 10 milligrams of gluten per day (Biagi et al., 2004).

If undiagnosed or untreated, CD has serious health consequences that maybe life threatening, particularly in infants. CD causes deformation ofabsorptive villae of the small intestine and may lead to destruction ofthe villi. As a result, nutrients are poorly absorbed and this may beassociated with weight loss, fatigue, mineral deficiencies, dermatitisand loss of night vision as well as intense intestinal distress whichusually includes bloating, diarrhea and cramps. Subjects with untreatedCD have increased risks of cancer such as a 10-fold increased risk ofcarcinoma of the small intestine, a 3-6 fold increase in the risk ofnon-Hodgkin lymphoma and 28-fold increased risk of intestinal T-celllymphoma. CD also presents a 3-fold increase in the risk of Type Idiabetes (Peters et al. 2003; Peters et al. 2003; Verkarre et al. 2004).A five fold increase in the incidence of mental depression has beenreported in coeliac patients (Pynnonen et al. 2004).

The molecular basis of coeliac disease is now reasonably well understood(Solid 2002; Hadjivassiliou et al. 2004) as being a reaction to aspecific sequence of amino acids in prolamins. Poorly digested prolaminpeptides rich in proline and glutamine conform to the substrate motiftargeted by human tissue trans-glutaminase (tTG) in the intestinalmucosa allowing key glutamine residues to be deamidated. The resultantnegatively charged glutamic acid allows the deamidated prolamin to bindto a specific class of HLA molecules (DQ2 or DQ8) (Kim et al. 2004).Specific T-cell clones, so called DQ2(8)/CD4⁺ restricted T-cells,targeted to the intestinal endothelium are stimulated to proliferate,releasing lymphokines which drive villous atrophy or antibody production(Hadjivassiliou et al. 2004). These T-cell clones reach a maximumconcentration in the peripheral blood of coeliacs around six days aftera dietary challenge (Anderson et al. 2000). The coeliac toxicity ofpurified proteins may therefore be sensitively and specificallydetermined by measuring their capacity to stimulate T-cells to produceIFN-γ, a cytokine fundamental to the pathogenesis of the enteropathyseen in coeliac disease. It therefore appears that the disease is causedby host's immune system reacting to prolamins as if they are an invadingpathogen, mounting a vigorous immune response, rather than as anallergy.

Wheat gluten is composed of many hundreds of different but relatedproteins including the monomeric gliadins and the polymeric glutenins.Gliadins account for about half of the gluten fraction and α-gliadinscomprise over 50% of the gliadins (Wieser et al., 1994; Gellrich et al.,2003). To date, the majority of coeliac toxicity data has focused onα-gliadin, the first prolamin to be cloned and fully sequenced (Kasardaet al. 1984). The coeliac toxicity of wheat α-gliadin is largelydetermined by a single glutamine residue within a key 17 amino acidepitope (Arentz-Hansen et al. 2000; Anderson et al. 2000; Shari et al.2002). Naturally occurring and synthetic peptides carrying pointmutations in this region have been identified which are not toxic (Vaderet al. 2003). Therefore, it appears likely that other non-toxic butfunctional prolamin molecules may be identified. At present, usefulprediction of coeliac toxicity is limited to the small fraction ofprolamins which have been characterized in terms of amino acid sequenceor the nucleotide sequence of the genes encoding them.

Barley is a diploid cereal that is widely grown in cooler climates forfood and beverage production. Barley seed proteins are classified intoalbumin, globulin, prolamin (hordein) and glutelin according to theirsolubility in water, salt solution, aqueous alcohol and basic or acidsolutions, respectively. Approximately half of the seed storage proteinsin barley are found in the prolamin fraction. These prolamins areprimarily reserve proteins that function as sources of carbon, nitrogenor sulphur for growth and development following germination. Hordeinconstitutes about 40% of the seed protein, although this is dependent onthe nitrogen supply of the plant during growth. The loci encoding thebarley prolamins have been characterized, mostly because of theircontribution to barley malting quality and foam formation and haze inbeer production. There are four classes of prolamins in barley, the B,C, D and γ-hordeins encoded by the Hor2, Hor1, Hor3, and Hor5 loci,respectively, on chromosome 1H (Shewry et al. 1999). These loci encodeproteins which vary from a single prolamin (e.g. D hordein) to proteinfamilies containing 20-30 members (e.g. B and C hordeins). The 13 and Chordeins are relatively more abundant, comprising about 70% and 24% ofthe total hordeins, respectively. The D and γ-hordeins represent minorcomponents at about 2-4% each. The molecular weight of hordeins variesfrom about 35 kDa to 100 kDa. There are no barley prolamins which haveclose homology to wheat α-gliadins, however it is widely accepted thathordeins are toxic to coeliacs (Williamson & Marsh 2000). The extent towhich the individual hordeins of barley are CD-inducing has not beenreported.

Beer is a widely consumed product made from malted barley, thereforebeer is widely assumed to be not suitable for coeliacs and generallyexcluded from their diet. Kanerva et al. (2005) were able to identifyprolamins at low levels in all but one of a number of beers. Physiciansand nutritionists generally urge their CD patients to assiduously avoidany wheat, barley or rye containing products, including beer. In the US,the FDA definition of “gluten free” requires the product to be made fromgluten-free raw materials only, i.e. containing no wheat, barley or ryewhatsoever. The Codex Alimentarius permits the “gluten-free” label onfoods containing no more than 200 ppm of gluten (0.2 g per kilogram orliter) and this is also the European standard for “gluten-free”. Mostcoeliacs can tolerate up to about 10 mg of gluten per day without majoreffect (Thompson, 2001).

Prolamins toxic to coeliac patients may be specifically detected withimmunoassays such as ELISA (Ellis et al., 1990; Sorell et al., 1998).These assays depend on the specific interaction between the protein andantibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and HPLC have also been used (Kanerva et al., 2005; Marchyloet al., 1986; Sheehan and Skerritt, 1997).

There is therefore a need for barley with substantially lower levels ofCD-inducing hordeins which could be used in food and drink products forCD-susceptible subjects.

SUMMARY OF THE INVENTION

There are four classes of prolamins in barley, the B, C, D andγ-hordeins encoded by the Hor2, Hor1, Hor3, and Hor5 loci, respectively.The present inventors have found that at least the B, C and D classesinduce undesirable inflammatory responses in subjects with coeliac'sdisease.

Whilst various barley mutants have previously been identified withproduce certain classes of hordeins at reduced levels, it had also beenobserved that this was at least compensated by the increased productionof other classes of hordeins. This suggests that the barley seed hascompensatory mechanisms to ensure certain levels of hordeins requiredfor the seed to be viable. Surprisingly, the present inventors havedetermined that most, if not all, of barley hordein production can beabolished and viable seeds obtained which are able to germinate andproduce barley plants in the field, despite the loss of the majorstorage form of nitrogen in the seed. These seeds are particularlyuseful for the production of foods and drinks for consumption bysubjects with coeliac's disease.

Thus, in one aspect the present invention provides a method of producinga food or malt-based beverage, the method comprising mixing barleygrain, or malt, flour or wholemeal produced from said grain, with atleast one other food or beverage ingredient, wherein the grain, malt,flour or wholemeal comprises about 25% or less of the level of hordeinswhen compared to grain from a corresponding wild-type barley plant ormalt, flour or wholemeal produced in the same manner from grain from acorresponding wild-type barley plant, thereby producing the food ormalt-based beverage.

Preferably, the grain, malt, flour or wholemeal comprises about 15% orless, about 10% or less, about 7.5% or less, about 5% or less or morepreferably about 2.5% or less of the level of hordeins when compared tograin of the corresponding wild-type barley plant, or wild-type malt,flour or wholemeal produced in the same manner.

Examples of a wild-type barley plant include, but are not limited to,Bomi, Sloop, Carlsberg II, K8 or L1.

In another embodiment, the grain comprises about 25% or less, about 20%or less, about 15% or less, about 10% or less, about 7.5% or less, about5% or less or more preferably about 2.5% or less of the level of B, Cand/or D hordeins or any combinations thereof when compared to grain ofthe corresponding wild-type barley plant. The malt, flour or wholemealmay comprise the same extent of reduction in the level of B, C and/or Dhordeins or any combinations thereof.

In another embodiment, the flour which comprises less than about 0.4%,less than about 0.3%, less than about 0.2% and more preferably less thanabout 0.1% hordeins. Hordein levels in flour produced from said graincan be determined by any technique known in the art such as alcoholfractionation.

In an embodiment, the grain has an average weight (100 grain weight) ofat least about 2.4 g. Preferably, the grain has an average weight ofabout 2.4 g to about 6 g, more preferably an average weight of about 3.5g to about 6 g.

In another embodiment, the starch content of the grain is at least about50%(w/w). More preferably, the starch content of the grain is about 50%to about 70%(w/w). The starch content can be determined using anytechnique known in the art. For example, a method as provided in Example4 can be used.

In a further embodiment, the coeliac toxicity of flour produced from thegrain is less than about 50%, less than about 25%, more preferably about10% or less, of flour produced from grain of a corresponding wild-typebarley plant. The coeliac toxicity may be determined using any techniqueknown in the art. For example, a method as provided in Example 1 can beused.

In yet another embodiment, malt produced from the grain comprises lessthan about 200 ppm hordeins, less than about 125 ppm hordeins, morepreferably less than about 75 ppm hordeins. The hordein content can bealso determined using any technique known in the art. For example, amethod as provided in Example 7 can be used.

In another embodiment, at least about 50% of the genome of the barleygrain is identical to the genome of a barley cultivar Sloop.

Preferably, the grain is from a plant which is homozygous for at leastone, at least two, at least three or more loci for a geneticvariation(s) which results in reduced levels of at least one, at leasttwo or all three hordein classes of the B, C and D classes when comparedto a corresponding wild-type barley plant. More preferably, thesegenetic variations are alleles which delete most or all of the B-hordeinencoding genes at the Hor2 locus and/or a mutant allele at the Lys3locus of barley.

In one embodiment, the grain is from a non-transgenic plant. Forexample, the grain can be from a cross between Riso 56 and Riso 1508 orprogeny thereof comprising the hor2 and lys3 mutations, respectively,present in these parental lines. Preferably, such progeny plantscomprise a substantially different genetic background to either Riso 56or Riso 1508, for example comprising less than about 25% of the geneticbackground of these parental lines.

In another embodiment, the grain is from a transgenic plant.

One embodiment of a transgenic plant is a plant that comprises atransgene which encodes a polynucleotide which down-regulates theproduction of at least one bordein in the grain. Preferably, thepolynucleotide of this embodiment is an antisense polynucleotide, asense polynucleotide, a catalytic polynucleotide, an artificial microRNAor a duplex RNA molecule which down-regulates expression of one orpreferably more genes encoding hordeins.

Another embodiment of a transgenic plant is a plant that comprises atransgene encoding a prolamin which is less toxic, or preferablynon-toxic, to a subject with coeliac's disease. An example of a prolaminwhich is non-toxic to a subject with coeliac's disease includes, but isnot limited to, oat avenins and maize zeins.

In an embodiment, the method comprises producing flour or wholemeal fromthe grain.

In a particularly preferred embodiment, the method further comprisesproducing malt from the grain. In an embodiment, the method furthercomprises fractionating dried germinated grain into two or more of anendosperm fraction, an endothelial layer fraction, a husk fraction, anacrospire fraction, and a malt rootlets fraction; and combining andblending predetermined amounts of two or more of the fractions.

With regard to the production of malt and beer, an important componentof the barley seed is starch. However, starch levels in barley mutantswith decreased levels of hordeins has previously been shown to havereduced levels of starch which would be expected to make the seedunsuitable for producing malt and beer. The inventors were particularlysurprised to find that barley seeds where most, if not all, of barleyhordein production had been abolished could be used to produce malt andbeer with suitable characteristics for commercial production. Thus, in aparticularly preferred embodiment, the malt-based beverage is beer orwhiskey, and the method comprises germinating the grain.

In an embodiment, the malt-based beverage is beer which comprises atleast about 2%, more preferably at least about 4%, alcohol. Preferably,the alcohol is ethanol.

In yet a further embodiment, the malt-based beverage is beer whichcomprises less than about 1 ppm hordeins.

In a further embodiment, at least about 50% of the grain germinateswithin 3 days following imbibition under typical conditions as used inmalting.

Examples of food products which can be produced using the methods of theinvention include, but are not limited to, flour, starch, leavened orunleavened breads, pasta, noodles, animal fodder, breakfast cereals,snack foods, cakes, malt, pastries or foods containing flour-basedsauces.

Preferably, the food or malt-based beverage is for human consumption. Ina further preferred embodiment, following consumption of the food ordrink at least one symptom of coeliac's disease is not developed by asubject with said disease.

In another aspect, the present invention provides a method of producinga food or malt-based beverage, the method comprising mixing maltcomprising one or more barley grain proteins and less than about 200 ppmhordeins and/or flour comprising one or more barley grain proteins andless than about 0.4% hordeins, at least one other food or beverageingredient thereby producing the food or malt-based beverage.

In an embodiment, the method comprises obtaining the malt and/or flour.

In yet another aspect, the present invention provides a method ofproducing a food or malt-based beverage, the method comprising mixingbarley grain, or malt, flour or wholemeal produced from said grain, withat least one other food or beverage ingredient, thereby producing thefood or malt-based beverage, wherein flour produced from the graincomprises less than about 0.4% hordeins, and/or malt produced from thegrain comprises less than about 200 ppm hordeins.

In an embodiment, the method comprises obtaining the malt and/or flour.

In another aspect, the present invention provides a barley plant whichproduces grain comprising about 25% or less of the level of hordeinswhen compared to grain from a corresponding wild-type barley plant.

Preferably, the grain comprises about 15% or less, about 10% or less,about 7.5% or less, about 5% or less or more preferably about 2.5% orless of the level of hordeins when compared to grain of thecorresponding wild-type barley plant.

Examples of a wild-type barley plant include, but are not limited to,Bomi, Sloop, Carlsberg II, K8 or L1.

In another embodiment, the grain comprises about 25% or less, about 20%or less, about 15% or less, about 10% or less, about 7.5% or less, about5% or less or more preferably about 2.5% or less of the level of B, Cand/or D hordeins or any combinations thereof when compared to grain ofthe corresponding wild-type barley plant.

In another embodiment, flour produced from the grain comprises less thanabout 0.4%, less than about 0.3%, less than about 0.2% and morepreferably less than about 0.1% hordeins.

In an embodiment, the grain has an average weight (100 grain weight) ofat least about 2.4 g. Preferably, the grain has an average weight ofabout 2.4 g to about 6 g, more preferably an average weight of about 3.5g to about 6 g.

In another embodiment, the starch content of the grain is at least about50%(w/w). More preferably, the starch content of the grain is about 50%to about 70%(w/w).

In a further embodiment, the coeliac toxicity of flour produced from thegrain is less than about 50%, less than about 25%, more preferably about10% or less, of flour produced from grain of a corresponding wild-typebarley plant.

In yet another embodiment, malt produced from the grain comprises lessthan about 200 ppm hordeins, less than about 125 ppm hordeins, morepreferably less than about 75 ppm hordeins.

In another embodiment, at least about 50% of the genome of the barleygrain is identical to the genome of a barley cultivar Sloop.

Preferably, the grain is from a plant which is homozygous for at leastone, at least two, at least three or more loci for a geneticvariation(s) which results in reduced levels of at least one, at leasttwo or all three hordein classes of the B, C and D classes when comparedto a corresponding wild-type barley plant.

In one embodiment, the grain is from a non-transgenic plant. Forexample, the grain can be from a cross between Riso 56 and Riso 1508 orprogeny thereof comprising the hor2 and lys3 mutations, respectively,present in these parental lines. Preferably, such grain comprises asubstantially different genetic background to either Riso 56 and Rise1508, for example comprising less than 25% of the genetic background ofthese parental lines.

In another embodiment, the grain is from a transgenic plant.

One embodiment of a transgenic plant is a plant that comprises atransgene which encodes a polynucleotide which down-regulates theproduction of at least one hordein in the grain. Preferably, thepolynucleotide of this embodiment is an antisense polynucleotide, asense polynucleotide, a catalytic polynucleotide, an artificial microRNAor a duplex RNA molecule which down-regulates expression of one orpreferably more genes encoding hordeins.

Another embodiment of a transgenic plant is a plant that comprises atransgene encoding a prolamin which is less toxic, preferably non-toxicto a subject with coeliac's disease. An example of a prolamin which isnon-toxic to a subject with coeliac's disease includes, but is notlimited to, oat avenin.

In a further embodiment, at least about 50% of the grain germinateswithin 3 days following imbibition under typical conditions as used inmalting.

In another aspect, the present invention provides a barley plant whichproduces grain, wherein flour produced from the grain comprises lessthan about 0.4% hordeins, and/or malt produced from the grain comprisesless than about 200 ppm hordeins.

In another aspect, the present invention provides grain of a barleyplant of the invention.

In a further aspect, the present invention provides a method ofproducing barley grain, the method comprising;

a) growing a barley plant of the invention,

b) harvesting the grain, and

c) optionally processing the grain.

Preferably, the plants are grown on a commercial scale in a field. Forexample, in one embodiment, the method comprises growing at least 1,000,more preferably at least 5,000, plants in a field in an area of at leastone hectare.

Also provided is a method of producing flour, wholemeal, starch or otherproduct obtained from grain, the method comprising;

a) obtaining grain of the invention, and

b) processing the grain to produce the flour, wholemeal, starch or otherproduct.

In a further aspect, the present invention provides a product producedfrom a barley plant of the invention or grain of the invention.

In an embodiment, the product is a food or malt-based beverage product.

Preferably, the malt-based beverage product is beer or whiskey.

In another embodiment, the product is a non-food product, preferablycomprising starch or consisting of at least about 50% starch. Examplesinclude, but are not limited to, films, coatings, adhesives, paper,building materials and packaging materials, or non-starch products suchas ethanol.

In yet another aspect, the present invention provides a food ormalt-based beverage produced using a method of the invention.

In an embodiment, the malt-based beverage is beer which comprises atleast about 2%, more preferably at least about 4%, alcohol. Preferably,the alcohol is ethanol.

In yet a further embodiment, the malt-based beverage is beer whichcomprises less than about 1 ppm hordeins.

In another aspect, the present invention provides beer comprising one ormore barley grain proteins and less than about 1 ppm hordeins. In anembodiment, the beer has less than about 0.05 ppm hordeins.

Preferably, the beer comprises at least about 2%, more preferably atleast about 4%, alcohol. Preferably, the alcohol is ethanol.

Examples of barley grain proteins include, but are not limited to, 9 kDalipid barley protein 1 (LTP1) and protein Z.

In another aspect, the present invention provides flour comprising oneor more barley grain proteins and less than about 0.4% hordeins.

In an embodiment, the flour comprises less than about 0.3%, less thanabout 0.2% and more preferably less than about 0.1% hordeins.

Preferably, the flour comprises less than about 7 mg, more preferablyless than about 5 mg, of alcohol soluble protein/gm dry weight flour.

In yet another aspect, the present invention provides malt comprisingone or more barley grain proteins and less than about 200 ppm hordeins.

In an embodiment, the malt comprises less than about 125 ppm hordeins,more preferably less than about 75 ppm hordeins.

In a further aspect, the present invention provides a method foridentifying barley grain which can be used to produce a food and/ormalt-based beverage for consumption by a subject with coeliac's diseasecomprising

a) obtaining one or more of the following materials;

-   -   i) a sample from a plant capable of producing said grain,    -   ii) the grain,    -   iii) malt produced from the grain, and/or    -   iv) an extract of said grain,

b) analysing the material from step a) for at least one hordein and/orat least one gene encoding a hordein,

wherein the greater the amount of hordeins produced by the grain theless suitable the grain is for producing a food and/or malt-basedbeverage for consumption by a subject with coeliac's disease.

In an embodiment, the sample is grain and step b) comprises analysingthe material for B and/or C hordeins. This can be performed using anytechnique known in the art, for example using an immunological methodsuch as ELISA assays. The method described in Example 1 can be used. Inan embodiment, step b) comprises orally administering the material fromstep a) to a subject with coeliac's disease and determining theimmunoreactivity of T cells obtained from the subject to one or morebarley hordeins.

In another embodiment, the sample material from step a) comprisesgenomic DNA and step b) comprises detecting the absence of one or morefunctional hordein genes. Again, this can be performed using anytechnique known in the art. For example, performing a gene amplificationstep as outlined in Example 9.

In an embodiment, the method comprises the step of selecting a barleyplant, grain or malt according to the invention from a plurality ofplants, grains or malts for propagation or use. Such selection is based,directly or indirectly, on the reduced coeliac toxicity of the material.

In a further aspect, the present invention provides a method ofpreventing or reducing the incidence or severity of coeliac's disease ina subject, the method comprising orally administering to the subject afood or malt-based beverage of the invention, or a grain of theinvention. Reduced incidence or severity of disease in this context isunderstood to be relative to administering an equal amount of food orbeverage prepared from wild-type barley. The food or beverage may beused to provide nutrients, or an increased amount of nutrients, to asubject having coeliac disease while lessening the risk of triggeringdisease symptoms.

In another aspect, the present invention provides for the use of a foodor malt-based beverage of the invention, or a grain of the invention,for the manufacture of a medicament for orally administering to asubject nutrients while at the same time preventing or reducing theincidence or severity of coeliac's disease.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to any other aspect of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1: Reverse-phase FPLC of total prolamin extracts, showing the A280nm chromatograms from wheat (a), barley (b), oats (c); maize (d) or ablank gradient (e). Prolamins equivalent to 0.2 g flour were loaded.Oxidised DTT is shown (DTT_(ox)); chromatograms have been offset forclarity.

FIG. 2: Reverse-phase FPLC of hordeins. A representative chromatogramshowing the A280 nm (solid line) and solvent composition (broken line)during isolation of hordein fraction 1 (#1), 2 (#2), 3 (#3), 4 (#4), 5(#5), or 6 (#6) from a barley extract. The indicated fractions werepooled as shown (bold line) from sequential injections.

FIG. 3: Analysis of 20 μg of hordein fractions #1-6 by SDS-PAGE,staining the gel with 0.06% Coomassie Blue 0250. The position ofmolecular weight standards (in kDa, BenchMark, Invitrogen) are indicatedin the left hand lane.

FIG. 4: The stimulation of IFN-γproduction in T-cells, isolated fromcoeliacs six days after a dietary challenge, by total prolaminpreparations from barley, wheat, oats or maize in the presence (●,n=21)), or absence (◯, n=13) of tTG pre-treatment. IFN-γ positivecolonies were counted and presented as mean SFU±S.E. Error bars are notshown when S.E. was smaller than symbols.

FIG. 5: The stimulation of IFN-γproduction in T-cells, isolated fromcoeliacs six days after a dietary challenge, by hordein fractions #1, 2,3, 4, 5, and 6, in the presence (●, n=21)), or absence (◯, n=13) of tTGpre-treatment. IFN-γpositive colonies were counted and presented as meanSFU±S.E. Error bars are not shown when S.E. was smaller than symbols.

FIG. 6: Analytical reverse-phase HPLC chromatograms of the isolatedhordein fractions. Representative chromatograms showing the A280 nmduring HPLC of hordein fractions #1, 2, 3, 4, 5, 6 purified from barley.For comparison, chromatograms are shown for wild-type barley (Himalaya)showing the elution (solid line) of D, C, and 13 hordeins, as well asthe mutant R56 which accumulates mainly C hordeins.

FIG. 7: Characterization of the prolamins in Riso56 and Riso1508 bySDS-PAGE and western blotting. Twenty μg of prolamin, purified as inExample 1, from the indicated barley line was incubated for 30 min atroom temperature in a buffer containing 6.6 M urea, 2% (w/v) SDS,1%(w/v) DTT, 62.5 mM Tris-HCl (pH 6.8), and 0.01% (w/v) bromophenol blueand loaded on duplicate 12% acrylamide gels and electrophoresed at 200 Vfor 40 min. The gel was rinsed in transfer buffer containing 192 mMGlycine, 25 mM Tris-base, and 20% (v/v) methanol for 10 min andtransferred to nitrocellulose (Amersham Hybond C+) at 100 V for 1 hr.The left hand membrane was stained in 0.2% (w/v) Ponceau S in 3% (w/v)trichloroacetic acid, 3% 5-sulphosalicylic acid and destained briefly inwater; the right hand membrane was blocked in 5% skim milk in PBST for 1hr, then incubated with mouse monoclonal antibody 12224 (Skerrit, 1988)in PBST, washed in PBST for 3×10 minutes, incubated in sheepanti-mouse-HRP (Selenius) in PBST, washed in PBST 3×5 min, incubated inAmersham ECL reagent as in the manufacturers instructions, and exposedto Amersham Hyperfilm. MAb 12224 was raised against a total gluteninextract and is detects all hordeins and prolamins (Skerrit, 1988).

FIG. 8: Reverse phase FPLC of hordein extracts in Riso56 and Riso1508compared to wild-type Bomi and Carlsberg II. Hordeins were purified fromthe indicated lines as in Example 1, and an amount equivalent to 0.2 gflour was injected onto an FPLC column using the first FPLC method inExample 1. The elution time of C-hordein (C-Hor) and B-hordein (B-Hor)is indicated.

FIG. 9: Representative SDS-PAGE of alcohol soluble proteins loaded on aper seed basis. Prolamin extracts (10 μl) from individual F2 barleyseeds from a cross between Riso1508 and Riso56 were extracted asdescribed above. The positions of protein standards of 30, 50, 70 and100 kDa are indicated on the left hand lane. The protein profiles of theparental lines Riso1508 and Riso56, and wild type (Bomi) are also shown.Six lanes from putative double nulls contain very little protein (null),six other lanes contain much reduced levels of protein (reduced).

FIG. 10: Representative SDS-PAGE of alcohol-soluble proteins loaded onan equal protein basis. Samples containing 20 μg of alcohol-solubleprotein extracted from individual F2 barley seeds were electrophoresedand the gel stained with Coomassie blue. Samples from the parental lines(Riso 1508 and Riso 56) and wild type (Bomi) are also run. The outermostand center lanes (10 kDa) contained protein standards of known molecularweights, the positions for bands of 30, 50, 70 and 100 kDa areindicated.

FIG. 11: RP-FPLC chromatograms of alcohol soluble extracts from F3barley seeds. Alcohol soluble proteins were extracted from individual F3seeds as described; the supernatants from two seeds were combined fromeach line and 50 μl injected onto a reverse phase FPLC column and elutedas described in Example 1.

FIG. 12: The content of water soluble (A), salt soluble (B), alcoholsoluble (C) and urea soluble (D) protein in duplicate flour samples fromwild type barley (Sloop, Carlsberg II, Bomi), the single null parents(Riso 56, Riso 1508) and F4 seeds of the plants of lines J4, J1, BB5,G1, 5RB was determined as in Example 4. The total extractable protein(E) content was determined by summing the content of the individualfractions. The total protein content was also estimated by elementalanalysis according to the method of Dumas (F). Protein contents areshown as the mean±SE.

FIG. 13: The coeliac toxicity of hordeins purified from various floursamples was determined with T-cells isolated from coeliacs, 6 days postchallenge, as in Example 5, and the mean spot forming units (SFU)±SEplotted vs the fresh weight of flour. For clarity, mean SFU are shownonly for hordeins purified from wild type barley (Sloop) or the doublenull line (G1) in the presence (+tTG) or absence (−tTG) of the enzyme,tissue transglutaminase (A). In all cases treatment with tTG increasedthe toxicity of hordeins as expected for coeliac disease. SFU are alsoshown for tTG treated hordeins (B) purified from flour samples ofwild-type barley (Sloop), the single null parents (R56, R1508) and F4seeds (4BH).

FIG. 14: Gene sequences specific for either the control gene(gamma3-Hor), or the B-hordein gene (B-Hor) were amplified from DNAextracts of individual F4 seedlings of either the lines 9RE, 4BH or theparent line R56 as in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in plant breeding,food technology, cell culture, molecular genetics, immunology, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology; A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal., (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan etal., (editors) Current Protocols in Immunology, John Wiley & Sons(including all updates until present).

As used herein, the term “barley” refers to any species of the GenusHordeum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. A preferred form of barley isthe species Hordeum vulgare.

Coeliac disease or celiac disease is an autoimmune disorder of the smallintestine that occurs in genetically predisposed individuals in all agegroups after early infancy. It affects approximately 1% of Indo-Europeanpopulations, though it is significantly underdiagnosed. Coeliac diseaseis caused by a reaction to gliadin, a gluten protein found in wheat (andsimilar proteins of Triticeae which includes other cultivars such asbarley and rye). Upon exposure to gliadin, the enzyme tissuetransglutaminase modifies the protein, and the immune systemcross-reacts with the bowel tissue, causing an inflammatory reaction.This leads to flattening of the lining of the small intestine, whichinterferes with the absorption of nutrients. The only effectivetreatment is a lifelong gluten-free diet. This condition has severalother names, including: coeliac disease (with ligature), c(o)eliacsprue, non-tropical sprue, endemic sprue, gluten enteropathy orgluten-sensitive enteropathy, and gluten intolerance. The symptoms ofcoeliac disease vary widely from person to person. Symptoms of coeliac'sdisease may include one or more of the following; gas, recurringabdominal bloating and pain, chronic diarrhea, constipation, pale,foul-smelling, or fatty stool, weight loss/weight gain, fatigue,unexplained anemia (a low count of red blood cells causing fatigue),bone or joint pain, osteoporosis, osteopenia, behavioral changes,tingling numbness in the legs (from nerve damage), muscle cramps,seizures, missed menstrual periods (often because of excessive weightloss), infertility, recurrent miscarriage, delayed growth, failure tothrive in infants, pale sores inside the mouth, called aphthous ulcers,tooth discoloration or loss of enamel, and itchy skin rash calleddermatitis herpetiformis. Some of the more common symptoms include;tiredness, intermittent diarrhoea, abdominal pain or cramping,indigestion, flatulence, bloating; and weight loss. Ceoliac's diseasecan be diagnosed, for example, as described in WO 01/025793.

As used herein, the term “non-toxic to a subject with coeliac's disease”refers to the consumption of food or a beverage not resulting in thedevelopment of a symptom of coeliac's disease in a subject sufferingfrom said disease. As described herein, the food or beverage made from acorresponding wild-type barley plant does result in disease symptoms.

The terms “seed” and “grain” are used interchangeably herein. “Grain”generally refers to mature, harvested grain but can also refer to grainafter processing such as, for example, milling or polishing, where mostof the grain stays intact, or after imbibition or germination, accordingto the context. Mature grain commonly has a moisture content of lessthan about 18-20%. Wild-type barley grain (whole grain) generallycontains 9-12% protein, and about 30-50% of this is prolamin, typically35%, so wild-type barley grain has about 3-4% prolamin by weight.Prolamin are found almost exclusively in the endosperm, which is about70% of the wholegrain weight.

As used herein, the term “corresponding wild-type” barley plant refersto a plant which comprises at least 50%, more preferably at least 75%,more preferably at least 95%, more preferably at least 97%, morepreferably at least 99%, and even more preferably 99.5% of the genotypeof a plant of the invention, but produces grain with unmodified hordeinlevels. In one embodiment, the “corresponding wild-type” barley plant isa cultivar used in plant breeding experiments to introduce geneticvariants that result in reduced hordein production in the grain. Inanother embodiment, the “corresponding wild-type” barley plant is aparental cultivar into which a transgene has been introduced whichreduces hordein production in the grain. In a further embodiment, the“corresponding wild-type” barley plant is a cultivar that is used at thedate of filing for the commercial production of barley grain such as,but not limited to, Bomi, Sloop, Carlsberg II, K8, L1, Vlamingh,Stirling, Hamelin, Schooner, Baudin, Gairdner, Buloke, WI3586-1747,WI3416, Flagship, Cowabbie, Franklin, SloopSA, SloopVic, Quasar, VB9104,Grimmett, Cameo*Arupo 31-04, Prior, Schooner, Unicorn, Harrington,Torrens, Galleon, Morex, Dhow, Capstan, Fleet, Keel, Maritime, Yarra,Dash, Doolup, Fitzgerald, Molloy, Mundah, Onslow, Skiff, Unicorn, Yagan,Chebec, Hindmarsh, Chariot, Diamant, Korál, Rubin, Bonus, Zenit, Akcent,Forum, Amulet, Tolar, Hens, Maresi, Landora, Caruso, Miralix, WikingettBrise, Caruso, Potter, Pasadena, Annabell, Maud, Extract, Saloon,Prestige, Astoria, Elo, Cork, Extract, Laura. In an embodiment, the“corresponding wild-type” barley plant produces grain with unmodifiedhordein levels due to it comprising a full complement of functionalhordein genes encoding functional hordein proteins, including the B, C,D and γ-hordeins encoded by the Hor2, Hor1, Hor3, and Hor5 loci.

As used herein, the term “one or more barley grain proteins” refers tonaturally occurring proteins produced by barley grain. Examples of suchproteins are known to those skilled in the art. Specific examplesinclude, but are not limited to, barley albumins such as the 9 kDa lipidtransfer protein 1 (LTP1) (see Douliez et al. (2000) for a review andSwiss-prot Accession No. P07597 as an example) and protein Z (see Brandtet al. (1990) and Genbank Accession No. P06293), including processed(mature) forms thereof, as well as denatured forms and/or fragmentsthereof produced as a result of the production of malt, flour,wholemeal, food or malt-based beverage of the invention.

As used herein, the term “malt” is used to refer to barley malt, “flour”to refer to barley flour, “wholemeal” to refer to barley wholemeal, and“beer” to refer to barley beer. More specifically, a source of malt,flour, beer, wholemeal, food product etc of the invention is from theprocessing (for example, milling and/or fermentation) of barley grain.These terms include malt, flout, beer, wholemeal, food product etcproduced from a mixture of grains. In a preferred embodiment, at least50% of the grain used to produce the malt, flour, beer, wholemeal, foodproduct etc is barley grain.

The term “plant” as used herein as a noun refers to a whole plant suchas, for example, a plant growing in a field for commercial barleyproduction. A “plant part” refers to plant vegetative structures (forexample, leaves, stems), roots, floral organs/structures, seed(including embryo, endosperm, and seed coat), plant tissue (for example,vascular tissue, ground tissue, and the like), cells, starch granules orprogeny of the same.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a gene construct (“transgene”) not foundin a wild-type plant of the same species, variety or cultivar. A“transgene” as referred to herein has the normal meaning in the art ofbiotechnology and includes a genetic sequence which has been produced oraltered by recombinant DNA or RNA technology and which has beenintroduced into the plant cell. The transgene may include geneticsequences derived from a plant cell. Typically, the transgene has beenintroduced into the plant by human manipulation such as, for example, bytransformation but any method can be used as one of skill in the artrecognizes.

“Nucleic acid molecule” refers to a polynucleotide such as, for example,DNA, RNA or oligonucleotides. It may be DNA or RNA of genomic orsynthetic origin, double-stranded or single-stranded, and combined withcarbohydrate, lipids, protein, or other materials to perform aparticular activity defined herein.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence, such as a polynucleotide definedherein, if it stimulates or modulates the transcription of the codingsequence in an appropriate cell. Generally, promoter transcriptionalregulatory elements that are operably linked to a transcribed sequenceare physically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising the proteincoding region of a structural gene and including sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof at least about 2 kb on either end and which are involved inexpression of the gene. The sequences which are located 5′ of the codingregion and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region which may be interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene which are transcribed intonuclear RNA (hnRNA); introns may contain regulatory elements such asenhancers. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide. The term“gene” includes a synthetic or fusion molecule encoding all or part ofthe proteins of the invention described herein and a complementarynucleotide sequence to any one of the above.

As used herein, the term “other food or beverage ingredient” refers toany substance suitable for consumption by an animal, preferably anysubstance suitable for consumption by a human. Examples include, but arenot limited to, water, grain from other plant species, sugar, etc.

As used herein, the term “genetic variation which each results inreduced levels of at least one hordein” refers to any polymorphism of abarley plant that reduces hordein production. The genetic variation maybe, for example, a deletion of a hordein gene(s) or part thereof, or amutation which reduce barley gene transcription. Examples of suchgenetic variations are present in Riso 56, Riso 527 and Riso 1508.Hence, such plants may be used for the methods of the invention.Furthermore, a plant of the invention may be a cross between any ofthese barley mutants. In a preferred embodiment, a plant of theinvention is a cross between Riso 56 and Riso 1508 or progeny thereofcomprising the hor2 and lys3 mutations present in these lines. In anembodiment, the plant is not a cross between Riso 527 and Riso 1508.

As used herein, unless stated to the contrary, the phrase “about” refersto any reasonable range in light of the value in question. In apreferred embodiment, the term “about” refers to +/−10%, more preferably+/−5%, of the specified value.

Prolamins and Hordeins

Cereal prolamins (known as gliadins in wheat, hordeins in barley,secalins in rye, avenins in oats, and zeins in maize) are the mainendosperm storage proteins in all cereal grains, with the exception ofoats and rice (Shewry and Halford, 2002). Hordeins represent 35-50% ofthe total protein in barley seeds (Jaradat, 1991). They are classifiedinto four groups, A (also known as γ hordein), B, C, and D, in order ofdecreasing mobility (Field et al., 1982). The B hordeins are the mainprotein fraction, differing from C hordeins in their sulphur content(Kreis and Shewry, 1989). B hordeins account for 70-80% of the total andC hordeins for 10-20% (Davies et al., 1993). The A hordeins are notgenerally considered to be a storage fraction whereas D hordeins arehomologous to the high-molecular-weight glutenins. Hordeins, along withthe rest of the related cereal prolamins, are not expressed in thezygotic embryo itself, unlike other storage proteins such as napins;they are believed to be expressed exclusively in the starchy endospermduring the middle-to-late stages of seed development.

Examples of barley hordein amino acid sequences (provided as AccessionNo; description in NCBI; gi details) include, but are not necessarilylimited to,

-   1103203A; hordein B; gi|224385|prf∥1103203A[224385];-   1103203B; hordein B; gi|224386|prf∥1103203B[224386]-   1103203C; hordein C; gi|224387|prf∥1103203C[224387]-   1210226A; hordein B1; gi|225171|prf∥1210226A[225171]-   1307151A; hordein C; gi|225588|prf∥1307151A[225588]-   1307151B; hordein C; gi|225589|prf∥1307151B[225589]-   1604464A; gamma hordein; gi|226755|prf∥1604464A[226755]-   AAA32942; C-hordein; gi|167016|gb|AAA32942.1|[167016]-   AAA32943; C-hordein storage protein;    gi|167018|gb|AAA32943.1|[167018]-   AAA32944; C-hordein storage protein;    gi|167020|gb|AAA32944.1|[167020]-   AAA32955; gamma-1 hordein precursor;    gi|167042|gb|AAA32955.1|[167042]-   AAA32967; hordein; gi|530093|gb|AAA32967.1|[530093]-   AAA92333; C hordein; gi|893242|gb|AAA92333.1|[893242]-   AAB28161; C-hordein [Hordeum vulgare];    gi|442524|gb|AAB28161.1∥bbm|324752|bbs|139926[442524]-   AAB71678; seed storage protein [Hordeum vulgare];    gi|2454599|gb|AAB71678.1|[2454599]-   AAB71679; seed storage protein [Hordeum vulgare];    gi|2454600|gb|AAB71679.1|[2454600]-   AAP31050; globulin [Hordeum vulgare];    gi|30421166|gb|AAP31050.1|[30421166]-   AAP31051; D-Hordein [Hordeum vulgare];    gi|30421167|gb|AAP31051.1|[30421167]-   AAQ63842; gamma 3 hordein [Hordeum chilense];    gi|34329251|gb|AAQ63842.1|[34329251]-   AAQ63843; gamma 3 hordein [Hordeum chilense];    gi|34329253|gb|AAQ63843.1|[34329253]-   AAQ63844; gamma 3 hordein [Hordeum chilense];    gi|34329255|gb|AAQ63844.1|[34329255]-   AAQ63845; gamma 3 hordein [Hordeum chilense];    gi|34329257|gb|AAQ63845.1|[34329257]-   AAQ63846; gamma 3 hordein [Hordeum chilense];    gi|34329259|gb|AAQ63846.1|[34329259]-   AAQ63847; gamma 3 hordein [Hordeum chilense];    gi|34329261|gb|AAQ63847.1|[34329261]-   AAQ63848; gamma 3 hordein [Hordeum chilense];    gi|34329263|gb|AAQ63848.1|[34329263] AAQ63849; gamma 3 hordein    [Hordeum chilense]; gi|34329265|gb|AAQ63849.1|[34329265]-   AAQ63850; gamma 3 hordein [Hordeum chilense];    gi|34329267|g|AAQ63850.1|[34329267]-   AAQ63851; gamma 3 hordein [Hordeum chilense];    gi|34329269|gb|AAQ63851.1|[34329269]-   AAQ63852; gamma 3 hordein [Hordeum chilense];    gi|34329271|gb|AAQ63852.1|[34329271]-   AAQ63853; gamma 3 hordein [Hordeum chilense];    gi|34329273|gb|AAQ63853.1|[34329273]-   AAQ63854; gamma 3 hordein [Hordeum chilense];    gi|34329275|gb|AAQ63854.1|[34329275]-   AAQ63855; gamma 3 hordein [Hordeum chilense];    gi|34329277|gb|AAQ63855.1|[34329277]-   AAQ63866; gamma 3 hordein [Hordeum chilense];    gi|34329299|gb|AAQ63866.1|[34329299]-   AAQ63867; gamma 3 hordein [Hordeum chilense];    gi|34329301|gb|AAQ63867.1|[34329301]-   AAQ63868; gamma 3 hordein [Hordeum chilense];    gi|34329303|gb|AAQ63868.1|[34329303]-   AAQ63869; gamma 3 hordein [Hordeum chilense];    gi|34329305|gb|AAQ63869.1|[34329305]-   AAQ63870; gamma 3 hordein [Hordeum chilense];    gi|34329307|gb|AAQ63870.1|[34329307]-   AAQ63871; gamma 3 hordein [Hordeum chilense];    gi|34329309|gb|AAQ63871.1|[34329309]-   AAQ63872; gamma 3 hordein [Hordeum chilense];    gi|34329311|gb|AAQ63872.1|[34329311]-   AAU06227; B hordein [Hordeum brevisubulatum subsp. turkestanicum];    gi|51556914|gb|AAU06227.1|[51556914]-   AAU06228; B hordein [Hordeum brevisubulatum subsp. turkestanicum];    gi|51556916|gb|AAU06228.1|[51556916]-   AAU06229; B hordein [Hordeum brevisubulatum subsp. turkestanicum];    gi|51556918|gb|AAU06229.1|[51556918]-   AAZ76368; B hordein [Hordeum vulgare subsp. vulgare];    gi|73427781|gb|AAZ76368.1|[73427781]-   ABA06537; B hordein [Hordeum vulgare subsp. vulgare];    gi|74422695|gb|ABA06537.1|[74422695]-   ABB82613; B hordein [Hordeum vulgare subsp. vulgare];    gi|82548223|gb|ABB82613.1|[82548223]-   ABB82614; B hordein [Hordeum vulgare subsp. vulgare];    gi|82548225|gb|ABB82614.1|[82548225]-   ABH01262; B hordein [Hordeum vulgare subsp. vulgare];    gi|110832715|gb|ABH01262.1|[110832715]-   BAA11642; D hordein [Hordeum vulgare subsp. vulgare];    gi|1167498|dbj|BAA11642.1|[1167498]-   CAA25509; unnamed protein product [Hordeum vulgare];    gi|18907|emb|CAA25509.1|[18907]-   CAA25912; unnamed protein product [Hordeum vulgare];    gi|18914|emb|CAA25912.1|[18914]-   CAA25913; unnamed protein product [Hordeum vulgare];    gi|829269|emb|CAA25913.1|[829269]-   CAA25914; unnamed protein product [Hordeum vulgare];    gi|18949|emb|CAA25914.1|[18949]-   CAA26889; unnamed protein product [Hordeum vulgare];    gi|18910|emb|CAA26889.1|[18910]-   CAA31861; unnamed protein product [Hordeum vulgare subsp. vulgare];    gi|18980|emb|CAA31861.1|[18980]-   CAA37729; B hordein precursor [Hordeum vulgare subsp. vulgare];    gi|18929|emb|CAA37729.1|[18929]-   CAA42642; unnamed protein product [Hordeum vulgare subsp. vulgare];    gi|9001|emb|CAA42642.1|[19001]-   CAA48209; D hordein [Hordeum vulgare subsp. vulgare];    gi|18970|emb|CAA48209.1|[18970]-   CAA51204; gamma 3 hordein [Hordeum vulgare];    gi|288709|emb|CAA51204.1|[288709]-   CAA59104; D-hordein [Hordeum vulgare subsp. vulgare];    gi|671537|emb|CAA59104.1|[671537]-   CAA60681; BI hordein [Hordeum vulgare];    gi|809031|emb|CAA60681.1|[809031]-   CAE45747; putative gamma 2 hordein [Hordeum vulgare];    gi|34365052|emb|CAE45747.1|[34365052]-   P06470; B1-hordein precursor; gi|123458|sp|P06470|HOR1_HORVU[123458]-   P06471; B3-hordein; gi|123459|sp|P06471|HOR3_HORVU[123459]-   P06472; C-hordein (PCP387); gi|123460|sp|P06472|HOR7_HORVU[123460]-   P17990; Gamma-hordein-1 precursor;    gi|123464|sp|P17990|HOG1_HORVU[123464]-   P17991; C-hordein (Clone PC HOR1-3);    gi|123461|sp|P17991|HOR8_HORVU[123461]-   P17992; C-hordein (Clone PC-919);    gi|123462|sp|P17992|HOR9_HORVU[123462]-   P29835; 19 kDa globulin precursor (Alpha-globulin);    gi|115505553|sp|P29835|GL19_ORYSJ[115505553]-   P80198; Gamma-hordein-3; gi|1708280|sp|P80198|HOG3_HORVU[1708280]

Examples of genes and/or cDNAs encoding barley hordeins (provided asAccession No; description in NCBI; gi details) include, but are notnecessarily limited to,

-   AF016237; Hordeum vulgare seed storage protein (HORDB3a) mRNA,    partial cds; gi|2454596|gb|AF016237.1|HVHORD1[2454596]-   AF016238; Hordeum vulgare seed storage protein (HORDB3a) mRNA 3′ end    sequence, partial cds; gi|2454597|gb|AF016238.1|HVHORD2[2454597]-   AH005570; Hordeum vulgare subsp. vulgare seed storage protein gene,    partial cds; gi|2454598|gb|AH005570.1|SEG_HVHORD[2454598]-   AJ580585; Hordeum vulgare gamma-2hor gene for putative gamma 2    hordein; gi|3436505|emb|AJ580585.1|[34365051]-   AY268139; Hordeum vulgare BAC 84G9, complete sequence;    gi|30421164|gb|AY268139.1|[30421164]-   AY338365; Hordeum chilense clone 1 cultivar H1 gamma 3 hordein mRNA,    complete cds; gi|34329250|gb|AY338365.1|[34329250]-   AY338366; Hordeum chilense clone 2 cultivar H1 gamma 3 hordein mRNA,    complete cds; gi|34329252|gb|AY338366.1|[34329252]-   AY338367; Hordeum chilense clone 3 cultivar H1 gamma 3 hordein mRNA,    complete cds; gi|34329254|gb|AY338367.1|[34329254]-   AY338368; Hordeum chilense clone 4 cultivar H1 gamma 3 hordein mRNA,    complete cds; gi|34329256|gb|AY338368.1|[34329256]-   AY338369; Hordeum chilense clone 5 cultivar H1 gamma 3 hordein mRNA,    complete cds; gi|34329258|gb|AY338369.1|[34329258]-   AY338370; Hordeum chilense clone 6 cultivar H1 gamma 3 hordein mRNA,    complete cds; gi|34329260|gb|AY338370.1|[34329260]-   AY338371; Hordeum chilense clone 7 cultivar H7 gamma 3 hordein mRNA,    partial cds; gi|34329262|gb|AY338371.1|[34329262]-   AY338372; Hordeum chilense clone 8 cultivar H7 gamma 3 hordein mRNA,    partial cds; gi|34329264|gb|AY338372.1|[34329264]-   AY338373; Hordeum chilense clone 9 cultivar H7 gamma 3 hordein mRNA,    partial cds; gi|34329266|gb|AY338373.1|[34329266]-   AY338374; Hordeum chilense clone 10 cultivar H7 gamma 3 hordein    mRNA, partial cds; gi|34329268|gb|AY338374.1|[34329268]-   AY338375; Hordeum chilense clone 11 cultivar H7 gamma 3 hordein    mRNA, partial cds; gi|3432920|gb|AY338375.1|[34329270]-   AY338376; Hordeum chilense clone 12 cultivar H7 gamma 3 hordein    mRNA, partial cds; gi|34329272|gb|AY338376.1|[34329272]-   AY338377; Hordeum chilense clone 13 cultivar H7 gamma 3 hordein    mRNA, partial cds; gi|34329274|gb|AY338377.1[34329274]-   AY338378; Hordeum chilense clone 14 cultivar H7 gamma 3 hordein    mRNA, partial cds; gi|34329276|gb|AY338378.1|[34329276]-   AY338379; Hordeum chilense clone 1 cultivar H47 gamma 3 hordein    gene, partial cds; gi|34329298|gb|AY338379.1[34329298]-   AY338380; Hordeum chilense clone 2 cultivar H47 gamma 3 hordein    gene, partial cds; gi|34329300|gb|AY338380.1|[34329300]-   AY338381; Hordeum chilense clone 3 cultivar H210 gamma 3 hordein    gene, partial cds; gi|34329302|gb|AY338381.1|[34329302]-   AY338382; Hordeum chilense clone 4 cultivar H210 gamma 3 hordein    gene, partial cds; gi|34329304|gb|AY338382.1|[34329304]-   AY338383; Hordeum chilense clone 5 cultivar H210 gamma 3 hordein    gene, partial cds; gi|34329306|gb|AY338383.1|[34329306]-   AY338384; Hordeum chilense clone 6 cultivar H210 gamma 3 hordein    gene, partial cds; gi|34329308|gb|AY338384.1|[34329308]-   AY338385; Hordeum chilense clone 7 cultivar H252 gamma 3 hordein    gene, partial cds; gi|34329310|gb|AY338385.1|[34329310]-   AY695367; Hordeum brevisubulatum subsp. turkestanicum B hordein    gene, complete cds; gi|51556913|gb|AY695367.1|[51556913]-   AY695368; Hordeum brevisubulatum subsp. turkestanicum B hordein    gene, complete cds; gi|51556915|gb|AY695368.1|[51556915]-   AY695369; Hordeum brevisubulatum subsp. turkestanicum B hordein    gene, complete cds; gi|51556917|gb|AY695369.1|[51556917]-   AY700807; Hordeum chilense cultivar H7 clone pC63-2 B3-hordein    pseudogene mRNA, complete cds; gi|57118094|gb|AY700807.1|[57118094]-   AY998005; Hordeum chilense clone pC39-1 D-hordein-like mRNA, partial    sequence; gi|66354246|gb|AY998005.1|[66354246]-   AY998008; Hordeum chilense clone pC36-2 (4) D-hordein-like mRNA,    partial sequence; gi|66354251|gb|AY998008.1|[66354251]-   AY998009; Hordeum chilense D-hordein gene, 5 UTR and partial cds;    gi|66354252|gb|AY998009.1|[66354252]-   AY998010; Hordeum chilense B-hordein gene, 5′ UTR and partial cds;    gi|66354254|gb|AY998010.1|[66354254]-   D82941; Hordeum vulgare Hor3 mRNA for D hordein, complete cds;    gi|1167497|dbj|D82941.1|BLYHOR3 [1167497]-   DQ148297; Hordeum vulgare subsp. vulgare cultivar XQ053 B hordein    gene, complete cds; gi|73427780|gb|DQ148297.1|[73427780]-   DQ178602; Hordeum vulgare subsp. vulgare cultivar Aba-siqing B    hordein gene, complete cds; gi|74422694|gb|DQ178602.1|[74422694]-   DQ189997; Hordeum vulgare subsp. vulgare clone Hn3 B hordein    pseudogene, complete sequence; gi|75991848|gb|DQ189997.1|[75991848]-   DQ267476; Hordeum vulgare subsp. vulgare clone Hn4 B hordein    pseudogene, complete sequence; gi|82548218|gb|DQ267476.1|[82548218]-   DQ267477; Hordeum vulgare subsp. vulgare clone Hn5 B hordein    pseudogene, complete sequence; gi|82548220|gb|DQ267477.1|[82548220]-   DQ267478; Hordeum vulgare subsp. vulgare clone Hn6 B hordein gene,    complete cds; gi|82548222|gb|DQ267478.1|[82548222]-   DQ267479; Hordeum vulgare subsp. vulgare clone Hn7 B hordein gene,    complete cds; gi|82548224|gb|DQ267479.1|[82548224]-   DQ267480; Hordeum vulgare subsp. vulgare clone Hn8 B hordein    pseudogene, complete sequence; gi|82548226|gb|DQ267480.1|[82548226]-   DQ267481; Hordeum vulgare subsp. vulgare clone Hn9 B hordein    pseudogene, complete sequence; gi|82548228|gb|DQ267481.1|[82548228]-   DQ826387; Hordeum vulgare subsp. vulgare B hordein gene, complete    cds; gi|110832714|gb|DQ826387.1|[110832714]-   J01237; barley b1 hordein mrna (partial);    gi|167002|gb|J01237.1|BLYB1HOR[167002]-   K03147; Barley (Hordeum vulgare L.) C-hordein mRNA, clone pHvE-c251;    gi|167015|gb|K03147.1|BLYCHORD2[167015]-   M23836; Hordeum vulgare hordein (hor2-1) mRNA, 3′ UTR;    gi|530091|gb|M23836.1|BLYHOR21A[530091]-   M23869; Hordeum vulgare B1 hordein mRNA, 3′ end;    gi|530092|gb|M23869.1|BLYHORDB1A[530092]-   M35610; Barley C-hordein storage protein, 3′ end;    gi|167017|gb|M35610.11BLYCHORDA[167017]-   M35611; Barley C-hordein storage protein, end;    gi|167019|gb|M35611.1|BLYCHORDB[167019]-   M36378; Barley gamma-1 hordein storage protein gene, complete cds;    gi|167041|gb|M36378.1|BLYG1HORDA[167041]-   M36941; Hordeum vulgare C-hordein gene, complete cds;    gi|167062|gb|M36941.1|BLYHORDCA[167062]-   S66938; C-hordein [Hordeum vulgare=barley, M564, Genomic, 2806 nt];    gi|442523|bbm|324747|bbs|139925|gb|566938.1|[442523]-   X01024; Barley mRNA fragment for B1 hordein;    gi|18906|emb|X01024.1|[18906]-   X01777; Barley mRNA fragment for B3-hordein;-   X01778; Barley mRNA fragment for B1-hordein;    gi|18908|emb|X01778.1|[18908]-   X01779; Barley mRNA fragment for C-hordein (pcP387);    gi|18948|emb|X01779.1|[18948]-   X03103; Barley gene for B1 hordein; gi|18909|emb|X03103.1|[18909]-   X13508; Barley gene for storage protein gamma-hordein;    gi|18979|emb|X13508.1|[18979]-   X53690; Hordeum vulgare DNA for B-Hordein (pcr31);    gi|18928|emb|X53690.1|[18928]-   X53691; H. vulgare DNA for B hordein (pcr47);    gi|18930|emb|X53691.1|[18930]-   X60037; H. vulgare hor1-17 gene for C-hordein;    gi|19000|emb|X60037.1|[19000]-   X68072; H. vulgare mRNA for D hordein; gi|18969|emb|X68072.1|[18969]-   X72628; H. vulgare mRNA for gamma 3 hordein, 3′ end;    gi|288708|emb|X72628.1|[288708]-   X84368; H. vulgare Hor3 gene; gi|671536|emb|X84368.1|[671536]-   X87232; H. vulgare B1 hordein gene; gi|809030|emb|X87232.1|[809030]

One embodiment of the present invention relates to transgenic barleyplants comprising a prolamin which is non-toxic to a subject withcoeliac's disease. As shown herein, examples of such a prolamin are anoat avenin and a maize zein. Examples of oat avenin amino acid sequences(provided as Accession No; description in NCBI; gi details) include, butare not necessarily limited to,

-   1411172A; avenin fast component N9; gi|226123|prf∥1411172A[226123]-   1502200A; prolamin; gi|226227|prf∥1502200A[226227]-   AAA32713; avenin; gi|166551|gb|AAA32713.1|[166551]-   AAA32714; avenin; gi|166553|gb|AAA32714.1|[166553]-   AAA32715; avenin; gi|166555|gb|AAA32715.1|[166555]-   AAA32716; avenin; gi|166557|gb|AAA32716.1|[166557]-   AAB23365; gamma 3 avenin, coeliac immunoreactive protein 2, CIP-2,    prolamin 2; gi|256082|gb|AAB23365.1∥bbm|240522|bbs|113745[256082]-   AAB32025; alcohol-soluble avenin-3=23.2 kda protein [Avena    sativa=oat, Narymsky 943, Peptide, 201 aa];    gi|693794|gb|AAB32025.1∥bbm|352847|bbs|156888[693794]-   ABD14148; avenin [Avena sativa];    gi|86610884|gb|ABD14148.1|[86610884]-   CAE85306; unnamed protein product [Avena sativa];    gi|39923008|emb|CAE85306.1|[39923008]-   CAE85351; unnamed protein product [Avena saliva];    gi|39923098|emb|CAE85351.1[39923098]-   P27919; Avenin precursor; gi|114720|sp|P27919|AVEN_AVESA[114720]-   P80356; Avenin-3 precursor (Prolamin);    gi|728937|sp|P803561AVE3_AVESA[728937]-   Q09095; Avenin-A (Gamma-4 avenin) (Prolamin) (Celiac immunoreactive    protein 1) (CIP-1); gi|75107163|sp|Q09095|AVEA_AVESA[75107163]-   Q09097; Avenin-F (Gamma-3 avenin) (Prolamin) (Celiac immunoreactive    protein 2) (CIP-2); gi|75107165|sp|Q09097|AVEF_AVESA[75107165]-   Q09114; Avenin-E (Alpha-2 avenin) (Avenin N9) (Prolamin) (Celiac    immunoreactive protein 3) (CIP-3);    gi|75107166|sp|Q091141AVEE_AVESA[75107166]-   S06211; avenin alpha-2—small naked oat (fragment);    gi|82325|pir∥S06211[82325]-   S07621; avenin gamma-3—small naked oat (fragment);    gi|2119756|pir∥S07621[2119756]-   S07622; avenin gamma-4—small naked oat (fragment);    gi|82327|pir∥S07622[82327]    Malting

A malt-based beverage provided by the present invention involves alcoholbeverages (including distilled beverages) and non-alcohol beverages thatare produced by using malt as a part or whole of their startingmaterial. Examples include beer, happoshu (low-malt beer beverage),whisky, low-alcohol malt-based beverages (e.g., malt-based beveragescontaining less than 1% of alcohols), and non-alcohol beverages.

Malting is a process of controlled steeping and germination followed bydrying of the barley grain. This sequence of events is important for thesynthesis of numerous enzymes that cause grain modification, a processthat principally depolymerizes the dead endosperm cell walls andmobilizes the grain nutrients, In the subsequent drying process, flavourand colour are produced due to chemical browning reactions. Although theprimary use of malt is for beverage production, it can also be utilizedin other industrial processes, for example as an enzyme source in thebaking industry, or as a flavouring and colouring agent in the foodindustry, for example as malt or as a malt flour, or indirectly as amalt syrup, etc.

In one embodiment, the present invention relates to methods of producinga malt composition. The method preferably comprises the steps of:

(i) providing grain of a barley plant of the invention,

(ii) steeping said grain,

(iii) germinating the steeped grains under predetermined conditions and

(iv) drying said germinated grains.

For example, the malt may be produced by any of the methods described inHoseney (Principles of Cereal Science and Technology, Second Edition,1994: American Association of Cereal Chemists, St. Paul, Minn.).However, any other suitable method for producing malt may also be usedwith the present invention, such as methods for production of specialtymalts, including, but limited to, methods of roasting the malt. Onenon-limiting example is described in Example 6.

Malt may be prepared using only grain produced from barley plants of theinvention or in mixtures comprising other grains.

Malt is mainly used for brewing beer, but also for the production ofdistilled spirits. Brewing comprises wort production, main and secondaryfermentations and post-treatment. First the malt is milled, stirred intowater and heated. During this “mashing”, the enzymes activated in themalting degrade the starch of the kernel into fermentable sugars. Theproduced wort is clarified, yeast is added, the mixture is fermented anda post-treatment is performed.

In another embodiment, wort compositions can be prepared from the malt.Said wort may be first and/or second and/or further wort. In general awort composition will have a high content of amino nitrogen andfermentable carbohydrates, mainly maltose. Typically, wort is preparedby incubating malt with water, i.e. by mashing. During mashing, themalt/water composition may be supplemented with additionalcarbohydrate-rich compositions, for example barley, maize or riceadjuncts. Unmalted cereal adjuncts usually contain no active enzymes,and therefore rely on malt or exogenous enzymes to provide enzymesnecessary for sugar conversion.

In general, the first step in the wort production process is the millingof malt in order that water may gain access to grain particles in themashing phase, which is fundamentally an extension of the maltingprocess with enzymatic depolymerization of substrates. During mashing,milled malt is incubated with a liquid fraction such as water. Thetemperature is either kept constant (isothermal mashing) or graduallyincreased. In either case, soluble substances produced in malting andmashing are extracted into said liquid fraction before it is separatedby filtration into wort and residual solid particles denoted spentgrains. This wort may also be denoted first wort. After filtration, asecond wort is obtained. Further worts may be prepared by repeating theprocedure. Non-limiting examples of suitable procedures for preparationof wort is described in Hoseney (supra).

The wort composition may also be prepared by incubating barley plants ofthe invention or parts thereof with one or more suitable enzyme, such asenzyme compositions or enzyme mixture compositions, for example Ultrafloor Cereflo (Novozymes). The wort composition may also be prepared usinga mixture of malt and unmalted barley plants or parts thereof,optionally adding one or more suitable enzymes during said preparation.In addition, prolyl-endopeptidase enzymes which specifically destroy thetoxic amino linkages involved in coeliac disease could be added duringthe fermentation of the wort to reduce the toxicity of the residualhordeins (De Angelis et al., 2007; Marti et al., 2005; Stepniak et al.,2006).

Grain Processing

Barley grain of the invention can be processed to produce a food ornon-food product using any technique known in the art.

In one embodiment, the product is whole grain flour (an ultrafine-milledwhole grain flour, such as an ultrafine-milled whole grain flour; awhole grain flour, or a flour made from about 100% of the grain). Thewhole grain flour includes a refined flour constituent (refined flour orrefined flour) and a coarse fraction (an ultrafine-milled coarsefraction).

Refined flour may be flour which is prepared, for example, by grindingand bolting cleaned barley. The Food and Drug Administration (FDA)requires flour to meet certain particle size standards in order to beincluded in the category of refined barley flour. The particle size ofrefined flour is described as flour in which not less than 98% passesthrough a cloth having openings not larger than those of woven wirecloth designated “212 micrometers (U.S. Wire 70)”.

The coarse fraction includes at least one of: bran and germ. Forinstance, the germ is an embryonic plant found within the barley kernel.The germ includes lipids, fiber, vitamins, protein, minerals andphytonutrients, such as flavonoids. The bran includes several celllayers and has a significant amount of lipids, fiber, vitamins, protein,minerals and phytonutrients, such as flavonoids. Further, the coarsefraction may include an aleurone layer which also includes lipids,fiber, vitamins, protein, minerals and phytonutrients, such asflavonoids. The aleurone layer, while technically considered part of theendosperm, exhibits many of the same characteristics as the bran andtherefore is typically removed with the bran and germ during the millingprocess. The aleurone layer contains proteins, vitamins andphytonutrients, such as ferulic acid.

Further, the coarse fraction may be blended with the refined flourconstituent. Preferably, the coarse fraction is homogenously blendedwith the refined flour constituent. Homogenously blending the coarsefraction and refined flour constituent may help reduce stratification ofthe particles by size during shipping. The coarse fraction may be mixedwith the refined flour constituent to form the whole grain flour, thusproviding a whole grain flour with increased nutritional value, fibercontent, and antioxidant capacity as compared to refined flour. Forexample, the coarse fraction or whole grain flour may be used in variousamounts to replace refined or whole grain flour in baked goods, snackproducts, and food products. The whole grain flour of the presentinvention (i.e.—ultrafine-milled whole grain flour) may also be marketeddirectly to consumers for use in their homemade baked products. In anexemplary embodiment, a granulation profile of the whole grain flour issuch that 98% of particles by weight of the whole grain flour are lessthan 212 micrometers.

In further embodiments, enzymes found within the bran and germ of thewhole grain flour and/or coarse fraction are inactivated in order tostabilize the whole grain flour and/or coarse fraction. It iscontemplated by the present invention that inactivated may also meaninhibited, denatured, or the like. Stabilization is a process that usessteam, heat, radiation, or other treatments to inactivate the enzymesfound in the bran and germ layer. Naturally occurring enzymes in thebran and germ will catalyze changes to compounds in the flour, adverselyaffecting the cooking characteristics of the flour and the shelf life.Inactivated enzymes do not catalyze changes to compounds found in theflour, therefore, flour that has been stabilized retains its cookingcharacteristics and has a longer shelf life. For example, the presentinvention may implement a two-stream milling technique to grind thecoarse fraction. Once the coarse fraction is separated and stabilized,the coarse fraction is then ground through a grinder, preferably a gapmill, to form a coarse fraction having a particle size distribution lessthan or equal to about 500 micrometers. In an exemplary embodiment, thegap mill tip speed normally operates between 115 m/s to 144 m/s, thehigh tip speed generates heat. The heat generated during the process andthe airflow lead to a decrease in the microbial load of the coarsefraction. In further embodiments, prior to grinding in a gap mill, thecoarse fraction may have an average aerobic plate count of 95,000 colonyforming units/gram (cfu/g) and an average coliform count of 1,200 cfu/g.After passing through the gap mill the coarse fraction may have anaverage aerobic plate count of 10,000 cfu/g and an average coliformcount of 900 cfu/g. Thus, the microbial load may be noticeably decreasedin the coarse fraction of the present invention. After sifting, anyground coarse fraction having a particle size greater than 500micrometers may be returned to the process for further milling.

In additional embodiments, the whole grain flour or the coarse fractionmay be a component of a food product. For example, the food product maybe a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, anEnglish muffin, a muffin, a pita bread, a quickbread, arefrigerated/frozen dough products, dough, baked beans, a burrito,chili, a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, aready to eat meal, stuffing, a microwaveable meal, a brownie, a cake, acheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll,a candy bar, a pie crust, pie filling, baby food, a baking mix, abatter, a breading, a gravy mix, a meat extender, a meat substitute, aseasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup,sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, to meinnoodles, an ice cream inclusion, an ice cream bar, an ice cream cone, anice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, anextruded snack, a fruit and grain bar, a microwaveable snack product, anutritional bar, a pancake, a par-baked bakery product, a pretzel, apudding, a granola-based product, a snack chip, a snack food, a snackmix, a waffle, a pizza crust, animal food or pet food.

In alternative embodiments, the whole grain flour or coarse fraction maybe a component of a nutritional supplement. For instance, thenutritional supplement may be a product that is added to the dietcontaining one or more ingredients, typically including: vitamins,minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices,probiotics, extracts, prebiotics and fiber. The whole grain flour orcoarse fraction of the present invention includes vitamins, minerals,amino acids, enzymes, and fiber. For instance, the coarse fractioncontains a concentrated amount of dietary fiber as well as otheressential nutrients, such as B-vitamins, selenium, chromium, manganese,magnesium, and antioxidants, which are essential for a healthy diet. Forexample 22 grams of the coarse fraction of the present inventiondelivers 33% of an individual's daily recommend consumption of fiber.Further, 14 grams is all that is needed to deliver 20% of an individualsdaily recommend consumption of fiber. Thus, the coarse fraction is anexcellent supplemental source for consumption of an individuals fiberrequirement. Therefore, in a present embodiment, the whole grain flouror coarse fraction may be a component of a nutritional supplement. Thenutritional supplement may include any known nutritional ingredientsthat will aid in the overall health of an individual, examples includebut are not limited to vitamins, minerals, other fiber components, fattyacids, antioxidants, amino acids, peptides, proteins, lutein, ribose,omega-3 fatty acids, and/or other nutritional ingredients.

In additional embodiments, the whole grain flour or coarse fraction maybe a fiber supplement or a component thereof. Many current fibersupplements such as psyllium husks, cellulose derivatives, andhydrolyzed guar gum have limited nutritional value beyond their fibercontent. Additionally, many fiber supplements have a undesirable textureand poor taste. Fiber supplements made from the whole grain flour orcoarse fraction may help deliver fiber as well as protein, andantioxidants. The fiber supplement may be delivered in, but is notlimited to the following forms: instant beverage mixes, ready-to-drinkbeverages, nutritional bars, wafers, cookies, crackers, gel shots,capsules, chews, chewable tablets, and pills. One embodiment deliversthe fiber supplement in the form of a flavored shake or malt typebeverage, this embodiment may be particularly attractive as a fibersupplement for children.

In an additional embodiment, a milling process may be used to make amulti-grain flour, multi-barley flour, or a multi-grain coarse fraction.For example, bran and germ from one type of barley may be ground andblended with ground endosperm or whole grain barley flour of anothertype of barley. Alternatively bran and germ of one type of grain may beground and blended with ground endosperm or whole grain flour of anothertype of grain. In an additional embodiment, bran and germ from a firsttype of barley or grain may be blended with bran and germ from a secondtype of barley or grain to produce a multi-grain coarse fraction. It iscontemplated that the present invention encompasses mixing anycombination of one or more of bran, germ, endosperm, and whole grainflour of one or more grains. This multi-grain, multi-barley approach maybe used to make custom flour and capitalize on the qualities andnutritional contents of multiple types of grains or barleys to make oneflour.

The whole grain flour of the present invention may be produced via avariety of milling processes. An exemplary embodiment involves grindinggrain in a single stream without separating endosperm, bran, and germ ofthe grain into separate streams. Clean and tempered grain is conveyed toa first passage grinder, such as a hammermill, roller mill, pin mill,impact mill, disc mill, air attrition mill, gap mill, or the like. Inone embodiment, the grinder may be a gap mill. After grinding, the grainis discharged and conveyed to a sifter. Any sifter known in the art forsifting a ground particle may be used. Material passing through thescreen of the sifter is the whole grain flour of the present inventionand requires no further processing. Material that remains on the screenis referred to as a second fraction. The second fraction requiresadditional particle reduction, Thus, this second fraction may beconveyed to a second passage grinder. After grinding, the secondfraction may be conveyed to a second sifter. Material passing throughthe screen of the second sifter is the whole grain flour of the presentinvention. The material that remains on the screen is referred to as thefourth fraction and requires further processing to reduce the particlesize. The fourth fraction on the screen of the second sifter is conveyedback into either the first passage grinder or the second passage grinderfor further processing via a feedback loop. In an alternative embodimentof the invention, the process may include a plurality of first passgrinders to provide a higher system capacity.

It is contemplated that the whole grain flour, coarse fraction and/orgrain products of the present invention may be produced by any millingprocess known in the art. Further, it is contemplated that the wholegrain flour, coarse fraction and/or grain products of the presentinvention may be modified or enhanced by way of numerous other processessuch as: fermentation, instantizing, extrusion, encapsulation, toasting,roasting, or the like.

Polynucleotides which Down-Regulate the Production of a Hordein

In one embodiment, grain of the invention, and/or used in the methods ofthe invention, is from a transgenic barley plant which comprises atransgene which encodes a polynucleotide which down-regulates theproduction of at least one hordein in the grain. Examples of suchpolynucleotides include, but are not limited to, antisensepolynucleotide, a sense polynucleotide, a catalytic polynucleotide, anartificial microRNA or a duplex RNA molecule. When present in the grain,each of these polynucleotides result in a reduction in hordein mRNAavailable for translation.

Antisense Polynucleotides

The term “antisense polynucletoide” shall be taken to mean a DNA or RNA,or combination thereof, molecule that is complementary to at least aportion of a specific mRNA molecule encoding a hordein and capable ofinterfering with a post-transcriptional event such as mRNA translation.The use of antisense methods is well known in the art (see for example,G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer(1999)). The use of antisense techniques in plants has been reviewed byBourque (1995) and Senior (1998). Senior (1998) states that antisensemethods are now a very well established technique for manipulating geneexpression.

An antisense polynucleotide in a barley plant of the invention willhybridize to a target polynucleotide under physiological conditions. Asused herein, the term “an antisense polynucleotide which hybridisesunder physiological conditions” means that the polynucleotide (which isfully or partially single stranded) is at least capable of forming adouble stranded polynucleotide with mRNA encoding a protein, such as abarley hordein under normal conditions in a barley cell.

Antisense molecules may include sequences that correspond to thestructural genes or for sequences that effect control over the geneexpression or splicing event. For example, the antisense sequence maycorrespond to the targeted coding region of the genes of the invention,or the 5′-untranslated region (UTR) or the 3′-UTR or combination ofthese. It may be complementary in part to intron sequences, which may bespliced out during or after transcription, preferably only to exonsequences of the target gene. In view of the generally greaterdivergence of the UTRs, targeting these regions provides greaterspecificity of gene inhibition.

The length of the antisense sequence should be at least 19 contiguousnucleotides, preferably at least 50 nucleotides, and more preferably atleast 100, 200, 500 or 1000 nucleotides. The full-length sequencecomplementary to the entire gene transcript may be used. The length ismost preferably 100-2000 nucleotides. The degree of identity of theantisense sequence to the targeted transcript should be at least 90% andmore preferably 95-100%. The antisense RNA molecule may of coursecomprise unrelated sequences which may function to stabilize themolecule.

Catalytic Polynucleotides

The term catalytic polynucleotide/nucleic acid refers to a DNA moleculeor DNA-containing molecule (also known in the art as a “deoxyribozyme”)or an RNA or RNA-containing molecule (also known as a “ribozyme”) whichspecifically recognizes a distinct substrate and catalyzes the chemicalmodification of this substrate. The nucleic acid bases in the catalyticnucleic acid can be bases A, C, G, T (and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence forspecific recognition of a target nucleic acid, and a nucleic acidcleaving enzymatic activity (also referred to herein as the “catalyticdomain”). The types of ribozymes that are particularly useful in thisinvention are the hammerhead ribozyme (Haseloff and Gerlach, 1988,Perriman et al., 1992) and the hairpin ribozyme (Shippy et al., 1999).

The ribozymes in barley plants of the invention and DNA encoding theribozymes can be chemically synthesized using methods well known in theart. The ribozymes can also be prepared from a DNA molecule (that upontranscription, yields an RNA molecule) operably linked to an RNApolymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNApolymerase. When the vector also contains an RNA polymerase promoteroperably linked to the DNA molecule, the ribozyme can be produced invitro upon incubation with RNA polymerase and nucleotides. In a separateembodiment, the DNA can be inserted into an expression cassette ortranscription cassette. After synthesis, the RNA molecule can bemodified by ligation to a DNA molecule having the ability to stabilizethe ribozyme and make it resistant to RNase.

As with antisense polynucleotides described herein, the catalyticpolynucleotides should also be capable of hybridizing a target nucleicacid molecule (for example an mRNA encoding a barley hordein) under“physiological conditions”, namely those conditions within a barleycell.

RNA Interference

RNA interference (RNAi) is particularly useful for specificallyinhibiting the production of a particular protein. Although not wishingto be limited by theory, Waterhouse et al. (1998) have provided a modelfor the mechanism by which dsRNA (duplex RNA) can be used to reduceprotein production. This technology relies on the presence of dsRNAmolecules that contain a sequence that is essentially identical to themRNA of the gene of interest or part thereof, in this case an mRNAencoding a polypeptide according to the invention. Conveniently, thedsRNA can be produced from a single promoter in a recombinant vector orhost cell, where the sense and anti-sense sequences are flanked by anunrelated sequence which enables the sense and anti-sense sequences tohybridize to form the dsRNA molecule with the unrelated sequence forminga loop structure. The design and production of suitable dsRNA moleculesfor the present invention is well within the capacity of a personskilled in the art, particularly considering Waterhouse et al. (1998),Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029, and WO01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded (duplex) RNA product(s) with homology tothe target gene to be inactivated. The DNA therefore comprises bothsense and antisense sequences that, when transcribed into RNA, canhybridize to form the double-stranded RNA region. In a preferredembodiment, the sense and antisense sequences are separated by a spacerregion that comprises an intron which, when transcribed into RNA, isspliced out. This arrangement has been shown to result in a higherefficiency of gene silencing. The double-stranded region may compriseone or two RNA molecules, transcribed from either one DNA region or two.The presence of the double stranded molecule is thought to trigger aresponse from an endogenous plant system that destroys both the doublestranded RNA and also the homologous RNA transcript from the targetplant gene, efficiently reducing or eliminating the activity of thetarget gene.

The length of the sense and antisense sequences that hybridise shouldeach be at least 19 contiguous nucleotides, preferably at least 30 or 50nucleotides, and more preferably at least 100, 200, 500 or 1000nucleotides. The full-length sequence corresponding to the entire genetranscript may be used. The lengths are most preferably 100-2000nucleotides. The degree of identity of the sense and antisense sequencesto the targeted transcript should be at least 85%, preferably at least90% and more preferably 95-100%. The RNA molecule may of course compriseunrelated sequences which may function to stabilize the molecule. TheRNA molecule may be expressed under the control of a RNA polymerase IIor RNA polymerase promoter. Examples of the latter include tRNA or snRNApromoters.

Preferred small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. Preferably, the target mRNA sequencecommences with the dinucleotide AA, comprises a GC-content of about30-70% (preferably, 30-60%, more preferably 40-60% and more preferablyabout 45%-55%), and does not have a high percentage identity to anynucleotide sequence other than the target in the genome of the barleyplant in which it is to be introduced, e.g., as determined by standardBLAST search.

microRNA

MicroRNA regulation is a clearly specialized branch of the RNA silencingpathway that evolved towards gene regulation, diverging fromconventional RNAi/PTGS. MicroRNAs are a specific class of small RNAsthat are encoded in gene-like elements organized in a characteristicinverted repeat. When transcribed, microRNA genes give rise tostem-looped precursor RNAs from which the microRNAs are subsequentlyprocessed. MicroRNAs are typically about 21 nucleotides in length. Thereleased miRNAs are incorporated into RISC-like complexes containing aparticular subset of Argonaute proteins that exert sequence-specificgene repression (see, for example, Millar and Waterhouse, 2005;Pasquinelli et al., 2005; Almeida and Allshire, 2005).

Cosuppression

Another molecular biological approach that may be used isco-suppression. The mechanism of co-suppression is not well understoodbut is thought to involve post-transcriptional gene silencing (PTGS) andin that regard may be very similar to many examples of antisensesuppression. It involves introducing an extra copy of a gene or afragment thereof into a plant in the sense orientation with respect to apromoter for its expression. The size of the sense fragment, itscorrespondence to target gene regions, and its degree of sequenceidentity to the target gene are as for the antisense sequences describedabove. In some instances the additional copy of the gene sequenceinterferes with the expression of the target plant gene. Reference ismade to WO 97/20936 and EP 0465572 for methods of implementingco-suppression approaches.

Nucleic Acid Constructs

Nucleic acid constructs useful for producing transgenic plants canreadily be produced using standard techniques.

When inserting a region encoding an mRNA the construct may compriseintron sequences. These intron sequences may aid expression of thetransgene in the plant. The term “intron” is used in its normal sense asmeaning a genetic segment that is transcribed but does not encodeprotein and which is spliced out of an RNA before translation. Intronsmay be incorporated in a 5′-UTR or a coding region if the transgeneencodes a translated product, or anywhere in the transcribed region ifit does not. However, in a preferred embodiment, any polypeptideencoding region is provided as a single open reading frame. As theskilled addressee would be aware, such open reading frames can beobtained by reverse transcribing mRNA encoding the polypeptide.

To ensure appropriate expression of the gene encoding an mRNA ofinterest, the nucleic acid construct typically comprises one or moreregulatory elements such as promoters, enhancers, as well astranscription termination or polyadenylation sequences, Such elementsare well known in the art.

The transcriptional initiation region comprising the regulatoryelement(s) may provide for regulated or constitutive expression in theplant. Preferably, expression at least occurs in cells of the seed.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter. Thesepromoters have been used to create DNA vectors that have been expressedin plants; see, e.g., WO 84/02913. All of these promoters have been usedto create various types of plant-expressible recombinant DNA vectors.

The promoter may be modulated by factors such as temperature, light orstress. Ordinarily, the regulatory elements will be provided 5′ of thegenetic sequence to be expressed. The construct may also contain otherelements that enhance transcription such as the nos 3′ or the ocs 3′polyadenylation regions or transcription terminators.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence, and can bespecifically modified if desired so as to increase translation of mRNA.For a review of optimizing expression of transgenes, see Koziel et al.(1996). The 5′ non-translated regions can also be obtained from plantviral RNAs (Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaicvirus, Alfalfa mosaic virus, among others) from suitable eukaryoticgenes, plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to the use of constructs wherein the non-translated region isderived from the 5 non-translated sequence that accompanies the promotersequence. The leader sequence could also be derived from an unrelatedpromoter or coding sequence. Leader sequences useful in context of thepresent invention comprise the maize Hsp70 leader (U.S. Pat. Nos.5,362,865 and 5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the chimeric vector to thepolynucleotide of interest. The 3 non-translated region of a recombinantDNA molecule contains a polyadenylation signal that functions in plantsto cause the addition of adenylate nucleotides to the 3′ end of the RNA.The 3′ non-translated region can be obtained from various genes that areexpressed in plant cells. The nopaline synthase 3′ untranslated region,the 3′ untranslated region from pea small subunit Rubisco gene, the 3′untranslated region from soybean 7S seed storage protein gene arecommonly used in this capacity. The 3′ transcribed, non-translatedregions containing the polyadenylate signal of Agrobacteriumtumor-inducing (Ti) plasmid genes are also suitable.

Typically, the nucleic acid construct comprises a selectable marker.Selectable markers aid in the identification and screening of plants orcells that have been transformed with the exogenous nucleic acidmolecule. The selectable marker gene may provide antibiotic or herbicideresistance to the barley cells, or allow the utilization of substratessuch as mannose, The selectable marker preferably confers hygromycinresistance to the barley cells.

Preferably, the nucleic acid construct is stably incorporated into thegenome of the plant. Accordingly, the nucleic acid comprises appropriateelements which allow the molecule to be incorporated into the genome, orthe construct is placed in an appropriate vector which can beincorporated into a chromosome of a plant cell.

One embodiment of the present invention includes the use of arecombinant vector, which includes at least transgene outlined herein,inserted into any vector capable of delivering the nucleic acid moleculeinto a host cell. Such a vector contains heterologous nucleic acidsequences, that is nucleic acid sequences that are not naturally foundadjacent to nucleic acid molecules of the present invention and thatpreferably are derived from a species other than the species from whichthe nucleic acid molecule(s) are derived. The vector can be either RNAor DNA, either prokaryotic or eukaryotic, and typically is a virus or aplasmid.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Transgenic Plants

Transgenic barley plants, as defined in the context of the presentinvention include plants (as well as parts and cells of said plants) andtheir progeny which have been genetically modified using recombinanttechniques to cause production of at least one polynucleotide and/orpolypeptide in the desired plant or plant organ. Transgenic plants canbe produced using techniques known in the art, such as those generallydescribed in A. Slater et al., Plant Biotechnology—The GeneticManipulation of Plants, Oxford University Press (2003), and P. Christouand H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons(2004).

In a preferred embodiment, the transgenic plants are homozygous for eachand every gene that has been introduced (transgene) so that theirprogeny do not segregate for the desired phenotype. The transgenicplants may also be heterozygous for the introduced transgene(s), suchas, for example, in F1 progeny which have been grown from hybrid seed.Such plants may provide advantages such as hybrid vigour, well known inthe art.

Four general methods for direct delivery of a gene into cells have beendescribed: (1) chemical methods (Graham et al., 1973); (2) physicalmethods such as microinjection (Capecchi, 1980); electroporation (see,for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat.Nos. 4,945,050 and 5,141,131); (3) viral vectors (Clapp, 1993; Lu etal., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms(Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. An illustrative embodiment of a method for delivering DNA intoZea mays cells by acceleration is a biolistics α-particle deliverysystem, that can be used to propel particles coated with DNA through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with corn cells cultured in suspension. A particle deliverysystem suitable for use with the present invention is the heliumacceleration PDS-1000/He gun is available from Bio-Rad Laboratories.

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain up to 1000 or more foci ofcells transiently expressing a marker gene. The number of cells in afocus that express the exogenous gene product 48 hours post-bombardmentoften range from one to ten and average one to three.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Method disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402,5,932,479, and WO 99/05265.

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions that influence the physiological state of therecipient cells and that may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. Nos. 5,177,010, 5,104,310,5,004,863, 5,159,135). Further, the integration of the T-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome.

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908); soybean (U.S.Pat. Nos. 5,569,834, 5,416,011); Brassica (U.S. Pat. No. 5,463,174);peanut (Cheng et al., 1996); and pea (Grant et al., 1995).

Methods for transformation of cereal plants such as barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447,PCT/US97/10621, U.S. Pat. Nos. 5,589,617, 6,541,257, and WO 99/14314.Preferably, transgenic barley plants are produced by Agrobacteriumtumefaciens mediated transformation procedures. Vectors carrying thedesired nucleic acid construct may be introduced into regenerable barleycells of tissue cultured plants or explants, or suitable plant systemssuch as protoplasts.

The regenerable barley cells are preferably from the scutellum ofimmature embryos, mature embryos, callus derived from these, or themeristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

Tilling

Plants of the invention can be produced using the process known asTILLING (Targeting Induced Local Lesions IN Genomes). In a first step,introduced mutations such as novel single base pair changes are inducedin a population of plants by treating seeds (or pollen) with a chemicalmutagen, and then advancing plants to a generation where mutations willbe stably inherited. DNA is extracted, and seeds are stored from allmembers of the population to create a resource that can be accessedrepeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cel I, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to L6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique.

TILLING is further described in Slade and Knauf (2005) and Henikoff etal., (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

EXAMPLES Example 1 Materials and Methods

Isolation and Purification of Prolamins

To isolate prolamins from cereals, whole-meal flour (10 g) was stirredfor 30 min at 25° C. in 200 ml of buffer containing 20 mMtriethanolamine-HCl (TEA), 1% (w/v) sodium ascorbate, 1% (w/v)polyethylene glycol (MW 6000; PEG6000) and 200 μl of plant proteaseinhibitor cocktail (Sigma #P9599), all adjusted to pH 8. The suspensionwas centrifuged at 7,000 g for 15 min and the pellet washed twice moreto remove proteins soluble in aqueous buffer. Prolamins in the washedpellet were dissolved in 40 ml of 50% (v/v) propan-2-ol containing 1%(w/v) dithiothreitol (DTT), 1% (w/v) PEG6000, 1% (w/v) sodium ascorbateby stirring for 30 min at 25° C. The suspension was centrifuged andprolamins precipitated from the supernatant with 2 volumes ofpropan-2-ol and stored at −20° C. When required, an aliquot equivalentto 10 g of flour was sedimented by centrifugation at 160 g at 4° C. for10 min, the pellet redissolved in 10 ml of buffer (buffer A) whichcontained 25 mM TEA, 8M freshly deionised urea and 1% DTT, all adjustedto pH 6, or other buffers as described.

A total prolamin fraction was purified from each grain sample by reversephase-fast protein liquid chromatography (RP-FPLC) as follows: Prolamins(200 μl) were injected into a 1 ml Resource RPC column (Pharmacia)connected in series with a similar 3 ml column. The column was washedwith 2 ml of 95% solvent A/5% solvent B and prolamins eluted with a 30ml linear gradient from 95% solvent A/5% solvent B to 100% solvent B at2 ml/min. Solvent A was 0.1% (v/v) trifluoroacetic acid (TFA) in water,solvent B was 0.1% (v/v) TFA in 60% (v/v) aqueous acetonitrile. Eluantcorresponding to protein peaks was pooled. Solvent controls weresimilarly pooled from runs without protein injection.

Barley hordeins were further fractionated by RP-FPLC as follows:Procedures were as above except that the elution gradient was varied sothat the concentration of solvent B was 50% at 4 ml, 52% at 17 ml, 56%at 34 ml, 58% at 37 ml, 60% at 41 ml, 62% at 44 ml, 64% at 47 ml, 66% at50 ml, 100% at 53 ml, 100% at 57 ml. One ml fractions were collected andfractions 11-14 (#1), 19-23 (#2), 31-34 (#3), 43-51 (#4), 53-58 (#5) and63-64 (#6) corresponding to A280 peaks were pooled.

Analytical Methods

Prolamin fractions were dissolved in 6M urea, 2% (w/v) SDS, 1% (w/v)DTT, 0.01% (w/v) bromophenol blue, 0.0625 M Tris-HCL (pH 6.8) at 25° C.and examined by SDS-PAGE as follows. A 5 μl aliquot of the prolamin-SDSsolution was loaded onto SDS-PAGE gels, using pre-cast 245×110×0.5 mm,8-18% polyacrylamide gradient gels (ExcelGel Pharmacia), and run at 600Vfor 90 min at 15° C. The gels were washed in 40% MeOH in 10% acetic acidfor 30 min, then water for 10 min. The prolamins were stained by soakingthe gel in 0.06% (w/v) colloidal Coomassie G250 in 8.5% phosphoric acidfor 30 min and the gels destained overnight in water. Each gel wascalibrated with a 10 kDa standard protein ladder (BenchMark,Invitrogen).

Hordein fractions were also dissolved in 50% (v/v) aqueous isopropylalcohol, 1% (w/v) DTT, treated with an excess of vinyl-pyridine toreduce di-sulphide bonds and examined by reverse phase-HPLC (RP-HPLC,Larroque et al., 2000) calibrated with prolamins isolated from barleylines Riso56 or Riso1508 where the entire 13 or C hordein families,respectively, are not accumulated due to mutation (Doll, 1983).

Protein levels in extracts or fractions were determined by the method ofBradford (1976). Typically, the protein content was measured in a96-well format by adding 10 μl of each DTT/propan-2-ol supernatant to200 μl of a 1 in 5 dilution of Coomassie protein assay concentrate(BioRAD) in water, calibrated against gamma globulin, and measuring theabsorbance at 595 nm.

Ex Vivo T-cell Toxicity Assays

Prolamins (50 mg/ml in 2M urea) were diluted with PBS containing 1 mMCaCl₂, to give either 62.5, 250, 625, 2500, or 6250 μg prolamin/ml anddeamidated by adding 25 μl of each solution to 100 μl of guinea pigliver tTG (transglutaminase (Sigma, T5398), 25 μg/ml tTG in PBScontaining 1 mM CaCl₂) and incubated for 6 hr at 37° C. Non-deamidatedsolutions were similarly prepared by incubation in the absence of tTG.Solvent controls were added as for the highest prolamin concentrations,Other controls contained either a known toxic ω-gliadin peptidedesignated 626fEE at 50 μg/ml, the 626fEE peptide alone or with tetanustoxoid (50 light forming units/ml), The ω-gliadin peptide 626fEE alsoknown as DQ2-ω-1 had the amino acid sequence QPEQPFPQPEQPFPWQP (SEQ IDNO:1) and was synthesised by Mimotopes, Melbourne, Australia. Itsidentity and purity (91%) were confirmed by mass spectrometry and HPLC.Tetanus toxoid was obtained from the Commonwealth Serum Laboratories,Melbourne. All solutions were then frozen at −20° C.

Twenty one, biopsy-proven HLA-DQ2⁺ coeliac disease subjects, who hadadhered to a strict gluten-free diet for at least three months, wereprovided 150 g of boiled barley daily for 3 days, consumed as part oftheir diet which otherwise remained gluten-free. Heparinised venousblood was collected either immediately prior to or six days aftercommencement of dietary challenge and peripheral blood mononuclear cells(PBMC) isolated by Ficoll-Hypaque density centrifugation (Anderson etal., 2000) from each blood sample. The PBMC cells were resuspended incomplete HT-RPMI medium (Invitrogen) containing 10% heat-inactivated,pooled, human AB serum. Deamidated or non-deamidated prolamins andcontrol solutions were thawed and 25 μl added to wells containing 100 μlof PBMC (3-8×10⁵ PBMC per well). These were cultured at 37° C. overnightin 96-well plates (MAIP-S-45; Millipore, Bedford, Mass.). Controlcultures were made by adding 25 μl of PBS containing 1 mM CaCl₂ (bufferalone controls). Final prolamin concentrations were 2.5, 10, 25, 100 or250 μg/ml and the final urea concentration was 50 mM. The level of IFN-γproduced in each culture, indicative of the toxicity of each prolamin,was assayed visually by spot formation using secondary antibodiesaccording to the suppliers instructions (Mabtech, Stockholm, Sweden) andspot forming units (SFU) counted using an automated ELISPOT reader (AIDAutoimmun Diagnostika GmbH; Germany). Results were presented as the meanspot forming units (SFU)±S.E. Typically, intra-assay percent coefficientof variation of SFU/10⁶ PBMC was 14% based on six duplicate assays of apositive control incubated with 0.5×10⁶ cells in six CD subjects (allwith >20 SFU/well).

Statistical Analysis

Analysis of variance (ANOVA) or t-tests using GenStat was used todetermine the significance of the differences observed for the mean SFUproduced by T-cells isolated from coeliac subjects either before (n=10)or after (n=21) a dietary challenge and incubated with hordeins,prolamins or controls.

The response curves for the 21 post-challenge individuals were verydifferent and a large proportion of the variability was due to thesedifferences. In order to take account of the different patient response,a random coefficients model was fitted. This is a mixed model analysisthat is performed using Residual Maximum Likelihood (REML) and whichallows for random terms involving the subject (patient) and thechallenge (the protein concentration) within patient. In order tostabilize the substantial heterogeneity of variance the data were logtransformed prior to this analysis. In order to deal with the problem ofzero counts one was added to all data prior to taking logs. The fixedterms in the model were the presence or absence of tTG and the hordeinfraction that was involved, together with their interaction.

A hyperbolic model was also fitted to the untransformed mean SFU forT-cells from the 21 post challenge patients, exposed to the six tTGhordein fractions or the four tTG treated cereal prolamin preparations.

Barley Transformation

Transformed barley plants may be produced by the method of Tingay etal., (1997). The gene constructs in binary vectors may be introducedinto a highly virulent Agrobacterium strain (AGL1) by tri-parentalconjugation, which is then used to introduce the T-DNA containing thetransgene and the selectable marker gene (encoding hygromycinresistance, expressed from the CaMV35S promoter) into regenerable cellsof the scutellum of immature barley embryos, as follows.

Developing barley seeds from the variety Golden Promise, 12-15 daysafter anthesis, are removed from the growing spike of greenhouse grownplants and sterilised for ten minutes in 20% (v/v) bleach followed byrinsing once with 95% ethanol and seven times with sterile water.Embryos (approx 1.5 to 2.5 mm in size) are then removed from the seedsunder aseptic conditions and the axis cut from each embryo. The embryosare placed cut side down on a petri dish containing callus inductionmedium. The Agrobacterium transconjugants are grown in MG/L broth(containing 5 g mannitol, 1 g L-glutamic acid, 0.2 g KH₂PO₄, 0.1 g NaCl,0.1 g MgSO₄.7H₂O, 5 g tryptone, 2.5 g yeast extract and 1 μg biotin perlitre, pH 7.0) containing spectinomycin (50 mg/L) and rifampicin (20mg/L) with aeration at 28° C. to a concentration of approximately2-3×10⁸ cells/ml. Then, approximately 300 μl of the cell suspension isadded to the embryos in a petri dish. After 2 min, excess liquid istipped from the plate and the embryos are flipped so that the cut side(axil side of the scutellum) is upwards. The embryos are thentransferred to a fresh plate of callus inducing medium and placed in thedark for 2-3 days at 24° C. The embryos are transferred to callusinducing medium with selection (50 μg/ml hygromycin and 15 μg/mltimentin).

Embryos remain on this media for 2 weeks in the dark at 24° C. Healthycallus is then divided and placed on fresh selection media and incubatedfor a further two weeks at 24° C. in the dark. Following this, theembryos are incubated at 24° C. in the light for 2 weeks on regenerationmedium containing cytokinin and transferred to rooting media containingcytokinin and auxin for three 2 week periods. Juvenile plants are thentransferred to soil mixture and kept on a misting bench for two weeksand finally transferred to a glasshouse.

Mutagenesis Methods Including Gamma Irradiation

Mutation of genes in barley leading to reduced expression of D, C, B orγ-hordeins can be achieved through either gamma ray irradiation orchemical mutagenesis, for example with ethyl methane sulfonate (EMS).For gamma ray induced mutation, seeds may be irradiated at a dose of20-50 kR from a ⁶⁰Co source (Zikiryaeva and Kasimov, 1972). EMSmutagenesis may be performed by treating the seeds with EMS (0.03%, v/v)as per Mullins at al. (1999). In a B+C double null background, mutantgrains may be identified on the basis of decreased protein or hordeincontent or altered grain morphology and confirmed by the methodsdescribed above. Mutants in one hordein gene can be crossed with asecond mutant to combine the mutations and produce a non-transgenicvariety of barley substantially lacking hordeins in the endosperm.

Example 2 Toxicity of Barley Hordeins to Coeliacs

Prolamin Composition of Barley and Other Cereals

Prolamins were isolated as aqueous-alcohol soluble proteins from thecoeliac toxic cereals, barley and wheat, the less toxic oats andnon-toxic maize and purified by one round of RP-FPLC as described inExample 1. The protein elution profiles of the prolamins as determinedby A_(280nm) in the RP-FPLC (FIG. 1) showed a series of partiallyresolved peaks due to individual proteins eluted by the steeplyincreasing solvent gradient. Fractions containing protein from 10purifications for each cereal were combined and lyophylised. The typicalyield of prolamin from various cereals (2 g) was: maize, 10 mg; oats, 23mg; barley, 73 mg; and wheat, 114 mg. The total prolamins from eachcereal were lyophilized and stored for testing in the ex vivo T-cellassay (below). Solvent controls were also prepared from the RP-FPLCprocedure.

The barley prolamins (hordeins) were also fractionated by RP-FPLC asdescribed in Example 1. The elution profile obtained duringfractionation in an initial experiment is shown in FIG. 2. Six peakswere obtained and the protein from each recovered. Corresponding pooledfractions from twenty sequential injections were combined andlypohylised. Typical yields from 4 g of whole-meal flour were: fraction1, 19 mg; fraction 2, 26 mg; fraction 3, 14 mg; fraction 4, 104 mg;fraction 5, 24 mg and fraction 6, 11 mg.

The identity of the hordeins in each fraction was established bySDS-PAGE as described in Example 1 and confirmed by analytical RP-HPLC.The results are shown in FIGS. 3 and 6. HPLC showed that fraction #1contained about 39% D hordein, which ran at 90 kDa on SDS-PAGE, andabout 61% C hordeins which ran at 47 and kDa on SDS-PAGE (FIG. 3, #1).Fraction #2 contained C hordeins as shown by both SDS-PAGE and HPLC.Fraction #3 contained a broad protein band which ran at about 45 kDa onSDS-PAGE but which resolved into 6 peaks on HPLC, corresponding to theelution of both C and B hordeins. The composition was estimated by HPLCas containing about 43% and 57% C and B hordeins, respectively.Fractions #4, 5, 6, contained B hordeins; these fractions may alsocontain a small amount of gamma-hordein. Two dimensional electrophoresisand tryptic mass fingerprinting of these hordein fractions did notproduce sufficient unique peptide fragments to unequivocally identifyindividual hordeins. This may be due to slight sequence variationsbetween the isolated hordeins and the sequences available in the databases. The fractionation in this experiment therefore resulted inenrichment for particular hordeins from barley but not completepurification. Further purification can be achieved by further rounds ofRP-FPLC or RP-FLPC combined with ion exchange methods.

Samples of each hordein fraction were treated or not treated with tTG,which converts glutamine residues in the proteins to glutamate, and thenlyophilized for use in the T-cell assays.

Toxicity Assays

T-cell assays using PBMC isolated from confirmed coeliac-diseasesubjects were carried out as described in Example 1 to establish thetoxicity of the total prolamin preparations and the hordein fractions.PBMC were isolated before and after dietary challenge with barley, andprolamin samples were either treated or not treated with tTG. T-cellsisolated from a subset of 10 coeliacs prior to a dietary challenge wereunresponsive to prolamins. Statistical analysis using ANOVA showed therewas no significant difference (P=0.77) between the mean number of IFN-γpositive spots for the highest concentrations of all tTG treatedprolamin, peptide or hordein fractions (group mean SFU±S.E. 1.52±0.18)and control cultures (mean SFU±S.E. 1.40±0.45). In contrast, theanalysis showed that pre-challenge T-cells reacted strongly (P<0.001) tothe positive control tetanus toxoid (mean SFU±S.E. 22.3±4.72) comparedto prolamins. This shows that the isolated T-cells were functional andcapable of reacting to a known toxin and confirms that there were fewprolamin reactive T-cells in the populations isolated before the dietarychallenge.

In contrast to the lack of response to prolamins before dietarychallenge, T-cells isolated after the dietary challenge were highlyreactive. T-cells isolated from 21 coeliacs, 6 days post challenge,responded strongly to tTG treated prolamins when compared to T-cellsfrom a subset (n=13) of this group exposed to non-deamidated prolamins.FIG. 4 shows that of the cereals, total barley prolamins induced thehighest number of SFU followed in decreasing order by prolamins fromwheat, oats and then maize (FIG. 4 panels A, B, C, D, respectively).Although maize prolamin did provoke a low dose-dependant T-cell responsein these assays, it normally does not provoke a response in dietarychallenges and is considered a coeliac-safe cereal. Intestinal digestionmay destroy epitopes present in whole maize prolamins which remainedintact in our assay and stimulated T-cells in vitro.

Of the hordein fractions, fractions #1, #2 and #3 produced highernumbers of SFU than hordein fractions #4, #5 and #6 (FIG. 5).

As the concentration of prolamin in the assays was increased, the numberof IFN-γ spots increased in a hyperbolic manner in a similar fashion tothe Michaelis-Menten enzyme kinetics often seen between an enzyme andits substrate (FIGS. 4 and 5), although it was not clear why thisoccurred for these cellular assays.

Each 96 well plate contained a number of internal positive and negativecontrols. There was a small but significant difference (P<0.001) betweenmean SFU when control cultures and the solvent controls were compared(control cultures SFU 2.75±0.67 and 1.49±0.24; solvent controls SFU2.64±0.23 and 2.75±0.23 in the absence and presence of tTGrespectively). Although statistically significant, these values werevery small compared to the post challenge SFU in the positive controlsor the prolamin containing assays. This confirmed that solventimpurities did not generate false positives. The positive controlpeptide 626fEE, gave a consistently high response (mean SFU±S.E.29.55±4.38 and 33.60±2.97 in the absence and presence of tTGrespectively). The lack of response of 626fEE to tTG was expected sincethis peptide was synthesised with a glutamate in the 10^(th) residue anddoes not require tTG treatment for toxicity. Addition of the solventcontrol did not significantly inhibit the response of the positive626fEE peptide (P=0.13), confirming that solvent impurities did notgenerate false negatives. The plate to plate consistency of the tetanustoxoid controls (P=0.193) confirmed that differences in T-cell responseto prolamins was not due to plate to plate variation, but reflected thediffering sensitivity of T-cell populations from different subjects.

The variation between different subjects to the same prolaminconcentration varied by as much as 200-fold. Therefore, a randomcoefficients REML model was fitted to the normalized SFU data and it wasfound that a model allowing for curvature in patient responses due tothe different concentrations of the protein gave a significantly betterfit (P<0.001) to the data than a model that fitted a single patientresponse regardless of concentration, with the deviance changing from1982.28 (1616 df) to 1640.91 (1613 df). The main effects due to tTG(P<0.001) and the prolamin fraction (P<0.001) were highly significantand there was no interaction between them. This confirmed that prolaminresponsive T-cells were induced in coeliac subjects six days after thedietary challenge with barley. The fitted means, on a log scale, for thenormalized SFU data were 1.613 (no tTG) and 2.026 (plus tTG) with astandard error of difference (SED) of 0.0527, confirming thatpretreatment with tTG had a significant effect on the responses. Thefitted means for hordein fractions #1-#6 were 1.903, 1.909, 1.956,1.693, 1.724 and 1.733 respectively with an SED of 0.0826. These resultsshow that the hordein fractions fall into two significantly differenttoxicity groups with hordein fractions #1, #2 and #3 forming a moretoxic group than hordein fractions #4, #5 and #6.

It was interesting to note that the most toxic hordein fractions elutedfirst from reverse phase FPLC and HPLC, and were therefore more polarthan the later eluting, less toxic fractions.

Conclusions

T-cells isolated from 21 coeliacs, 6 days post challenge, respondedstrongly to tTG treated prolamins as compared to non-deamidatedprolamins as expected for coeliac disease (Hadjivassiliou et al., 2004;Kim et al., 2004). This could be explained by an interaction between thedeamidated prolamin and a binding site in a key protein, such as theHLA-DQ2 molecule, which presented stimulatory proteins to receptors onCD4⁺ T-cells involved in the inflammatory response.

Although there were measurable differences in the toxicity of hordeinfractions, all hordeins were significantly more toxic than maize and oatprolamin, which are regarded as safe for most coeliacs. The statisticalanalyses showed that the barley prolamin and hordein fractions #1, #2and #3 (containing D and C hordeins) formed the most toxic group.Hordein fractions #4, #5 and #6, containing mainly B hordeins, and wheatprolamins formed a second, less toxic group. Oats and maize prolaminsformed the least toxic group. This indicated that T-cells induced incoeliacs by a barley challenge were less sensitive to wheat and oats.This may be because the dominant epitopes in barley prolamins differconsiderably from those in wheat and oats prolamins. Although the fitteddata indicated that oat prolamins were significantly less toxic thanthose from barley, there was a fifty fold variation between differentsubjects to the same concentration of oat prolamin, with T-cells fromfive of the 21 subjects having over 20 SFU at the highest prolaminconcentration. This was consistent with other reports of individualswith serious coeliac responses to oats (Arentz-Hansen et al., 2004;Lundin et al., 2003).

It was considered likely based on this data that, in a dietarychallenge, all of the hordein fractions would provoke a significantintestinal reaction in coeliacs. This suggested that all hordeinfractions would need to be deleted or modified to produce barley whichwas completely non-toxic to coeliacs. It also suggested that hordeins Band C, the major components, should be removed or modified first of all.

Example 3 Production of Barley Grain Reduced for Both B and C Hordeins

A number of barley mutants affected in hordein synthesis or accumulationhave been identified previously. These barley mutants were not isolatedfor the purpose of reducing hordeins in the grain, but were isolated andselected for increased lysine levels in the grain and subsequently foundto be reduced for hordeins.

The mutant Riso 7, first described by Doll et al. (1976), was identifiedafter fast neutron treatment of the parent Bomi. It contained arecessive mutation in a gene that resulted in a 29% decrease inprolamins and a 10% increase in the lysine content of protein relativeto Bomi. The reduction in the lysine-poor prolamins was compensated forby an increase in other, relatively lysine-rich storage proteins,resulting in elevated lysine content. The grain yield and starch contentwere reduced by 6% and 7%, respectively, compared to the parent(Talberg, 1982; Doll, 1983).

Riso 56, first described by Don et al. (1973), was created by gamma-raymutation of the parent Carlsberg II. Kernel size, grain yield, andprolamin content were decreased by 30%, 47%, and 25%, respectively,relative to the parent while the lysine content of protein in the mutantgrain was increased by 13% compared to the parent. The decreased hordeincontent was associated with increased non-protein nitrogen and water andsalt-soluble proteins (Shewry P R et al. 1980). The high lysine contentof proteins in Riso 56 was due to a recessive mutation on chromosome 5(Ullrich and Eslick, 1978) at a genetic locus designated Hor2ca (Doll,1980). The mutation included deletion of 80-90 kb of DNA from the Hor2locus which encoded the B hordeins in barley. Expression of B hordeinproteins was reduced by 75% in the mutant while expression of the Chordeins was increased by 2-fold (Kreis et al., 1983). The deletion wasnot related to the translocation between chromosome 2 and 5 that wasalso present in Riso 56 (Olsen, 1977).

Rise 527, first described by Doll et al. (1973), was also created bygamma-ray mutation but from the parent Bomi. Kernel size, grain yieldand grain prolamin content were decreased by 13%, 25%, and 20%,respectively, relative to the parent while the lysine content of proteinin the mutant was increased by 12%. The mutation was recessive, in agene on chromosome 6 designated lys6i (Jensen, 1979). This mutant haddecreased levels of D hordeins and increased levels of B1 hordeins(Klemsdal et al., 1987).

Riso 1508 was identified after EMS mutation of the parent Bomi (Doll etal., 1973; Ingerversen et al., 1973; Doll, 1973). Kernel size, grainyield and grain prolamin content were decreased by 8%, 12%, and 70%,respectively, relative to the parent grain while the lysine content ofprotein in the mutant was increased by 42%. The high lysine content wasdue to a recessive mutation in a gene located near the centromere regionof barley chromosome 7 (Karlsson, 1977). This gene was first designatedas shrunken endosperm xenia sex3c (Ullrich and Eslick, 1977) but is nowgenerally known as lys3a (Tallberg, 1977). The relative levels ofprotein types in the mutant grain was changed, with more water solubleprotein (albumin/globulins) increased from 27% to 46% of total seedprotein nitrogen and less prolamin, decreased by 70% relative to theparent, from 29% to 9% of total seed protein nitrogen (Ingerversen etal., 1973; Doll, 1973). There was a four-fold increase in both freeamino-acids and non-protein N in Rise 1508, compared to the parent whenplants were grown under high levels of nitrogen fertilizer (Koeie andKreis, 1978). Shewry et al. (1978) confirmed that the level ofsalt-soluble non-protein nitrogen was doubled. The proportion of seednitrogen as hordein was decreased by 70% and the salt soluble proteinsincreased by 70% in Riso 1508 compared to Bomi. Detailed molecularanalysis showed that the levels of B and C hordeins were reduced by 80%and 93%, respectively, while the D hordeins were increased four-fold.These effects on protein accumulation were due to changes in mRNAabundance or stability (Kreis et al., 1984). This might have beenmediated by increased methylation of the promoters of the genes encodingthe B and C hordeins in the Riso 1508 mutant (Sorensen et al., 1996).The smaller seed size of Riso 1508 was mainly due to reduced synthesisof starch (Koeie and Breis, 1978; Kreis and Doll, 1980; Doll, 1983).Sugars were increased by two-fold while starch synthesis was decreasedby about 20-30% in Riso 1508 compared to the parent. Kreis (1979)reported that β-amylase levels were reduced in Riso 1508 while Hejgaardand Boisen (1980) reported similar levels of β-amylase.

Hiproly was a spontaneous mutant identified from Ethiopian germplasm CI3947 (Munck et al., 1970) which had increased levels of both totalprotein and protein lysine, increased by 20-30% relative to wild-typebarleys (Doll, 1983). When crossed to wild-type barley, the high proteincontent was lost while the increased protein lysine content wasretained, demonstrating that these traits were inherited independently.The increased lysine content was due to a single recessive mutation inthe lys gene on chromosome 7. The mutation increased the level of waterand salt soluble proteins and thereby the lysine content. Unlike theRiso high lysine mutants, the hordein levels and seed weight in Hiprolywere not decreased in backcrossed progeny. Non-protein nitrogen was alsonot increased. The content of β-amylase was increased 4-fold (Hejgaardand Boisen, 1980),

Characterisation of the Parental Lines Riso 56 and Riso 1508

The characteristics of prolamins accumulated by the parental lines Riso56 and Riso 1508 were confirmed by SDS-PAGE and reverse phase HPLC.Salt-soluble proteins extracted from grain were separated by gelelectrophoresis and transferred to membranes (Western blotting). Theprotein patterns on membranes stained for total protein (FIG. 7, lefthand side) or treated with a prolamin specific monoclonal antibody(mouse monoclonal antibody MAb12224, raised against a total gluteninextract, and which detects all hordeins and prolamins (Skerritt, 1988)(right hand side) showed that the levels of B hordeins were very low inRiso 56 while the C hordeins were increased relative to the levels inRiso 527. Antibody detection confirmed that the level of B hordeins inthe Riso 56 extract were extremely low (dotted box). The three proteinsseen in Riso 56 which co-migrated with the B hordeins were most likelyγ-hordeins. In Riso 1508, accumulation of the B hordeins was reducedwhile the C-hordeins were barely detectable (dotted box). This wasconsistent with the published literature. Levels of D hordein, which wasa relatively minor prolamin component, did not appear to be increased atthe protein loadings used in this gel.

FIG. 8 shows the relative levels of the different hordeins in purifiedextracts after reverse-phase FPLC analysis. Hordein extracts equivalentto 0.2 g of flour were analysed by FPLC as described in Example 1.Therefore, the area under the A280 chromatograms was proportional to therelative protein content of each sample. In Riso 56, levels of the Chordeins were increased by 400% and of the B hordeins decreased by 86%compared to the parent Carlsberg II. In Riso 1508, C and B hordeins wereboth reduced (91% and 86%, respectively) compared to the parent Bomi.These patterns were similar to the published data.

Identification of Seeds Having Both Hordein Mutations

Plants of the lines Riso 56 and Riso 1508 were crossed by emasculatingRiso 1508 and two days later pollinating them with fresh Riso 56 pollen.Ten F1 seeds were germinated and F1 plants grown and allowed toself-fertilize F2 seeds were harvested at maturity.

To identify double mutants in the population, half of each of 288 F2seeds were individually crushed and ground to a powder in a plasticmicrotube with a stainless-steel ball, shaken at 30/sec for 3×1.5 min ina 96 well Vibration Mill (Retsch Gmbh, Rheinische). An aliquot (400 μl)of an aqueous buffer was added to each tube to extract water solubleproteins. The buffer contained 20 mM triethylamine-HCl (TEA), 1% (w/v)sodium ascorbate, 1% (w/v) PEG6000 and 1/1000 dilution of plant proteaseinhibitor (Sigma P9599), pH 8 at room temperature (RT). The contents ofeach tube were shaken again and then centrifuged at 160 g for 10 min atRT. The water-insoluble flour pellet was washed twice more in the samemanner and respective supernatants pooled to give the water solublefractions. Alcohol soluble prolamins in the pellet were then extractedby adding 400 μl of 50% (v/v) aqueous propan-2-ol containing 1% (w/v)OTT and shaking the tubes as above, followed by incubation for 30 min atRT, a second round of shaking and centrifugation as above. Respectivesupernatants containing extracted prolamins were pooled and transferredto fresh tubes. The protein content in DTT/propan-2-ol supernatants wasmeasured with Coomassie reagent (BioRAD) and the prolamins in a 200 μlaliquot precipitated with 4000 of propan-2-ol and stored overnight at−20° C.

An aliquot of each prolamin half-seed extract was examined for the lossof B and C hordeins by SDS-PAGE as described in Example 1 (FIG. 9). Thescreening gels were loaded on a per seed basis, with each lane carryingthe equivalent of 1/20 of a seed. In particular, extracts were examinedfor the absence or reduction of the characteristic hordein protein bandsat 40 kDa (B hordein specific) and 70 kDa (C hordein specific). Seeds ofthe parental lines Riso 56 and Riso 1508 were reduced for B and Chordeins, respectively, but still contained low levels of D hordeins at100 kDa (FIG. 9). The majority of the F2 seed extracts contained awild-type pattern with D, C and B hordeins present (FIG. 9), confirmingthat an effective cross between the two parental lines had been made.Sixteen seeds appeared to lack both B and C hordeins and were thereforescored as homozygous for both of the genetic lesions present in theparents. These were identified from 288 half-seeds (frequency 0.055).This was similar to the frequency of 1 in 16 (0.0625) expected for thecombination of two simple, recessive mutations.

The total protein levels in the alcohol-soluble extracts of the F2 halfseeds were compared to those from wild-type and parental seeds. The dataare shown in Table 1. The protein levels in the extracts of the F2 seedswere reduced to less than 20%, in some cases less than 15% of thewild-type. These values may have been inflated by non-protein nitrogencompounds such as free amino-acids present in the extracts.

TABLE 1 Protein levels in alcohol-soluble extracts of F2 barley halfseeds. Alcohol soluble protein Sample (μg/seed ± SE) % Bomi ControlsBomi  512 ± 130 100%  Riso 56 364 ± 44 71% Riso 1508 147 ± 26 28% Doublenulls RE9 129.6 25% RF8 89.6 18% RH2 85.6 17% BA9 85.6 17% RB10 85.6 17%RA9 82.4 16% RG12 75.2 15% BB11 72.8 14% BD5 72 14% BD9 73.6 14% BE858.4 11% BF8 59.2 12% BB5 57.6 11% RB5 57.6 11%

The observed differences in prolamin levels between the F2 lines mayhave been due to the segregation of other genes or mutations from theparents.

Additional protein gels were run by taking a volume of theDTT/propan-2-ol supernatant containing 20 μg protein, drying each undervacuum in a SpeediVac, dissolving the protein in 20 μl of a buffercontaining 62.5 mM Tris-HCl (pH 6.8), 12.5% (w/v) glycerol, 2% (w/v)SDS, 1% (w/v) DTT, and 0.112% (w/v) bromophenol blue, and heating in aboiling water bath for 90 sec. Each solution was loaded on a precastSDS-polyacrylamide gel, electrophoresed, stained and examined asdescribed above. A typical gel is shown in FIG. 10. Most of the selectedF2 seeds appeared to lack both the B and C hordeins and were presumed tobe “double nulls”. Even though each lane was loaded with the same amountof protein as measured by the dye-binding protein assay, most of theextracts from the double nulls appeared to contain substantially lessprotein than the controls, in particular they contained littleproteinaceous material larger than 20 kDa. This might be explained bythe presence of non-protein nitrogen compounds such as free amino-acidsin the extracts which could have inflated the apparent protein levels asestimated by the dye-biding protein assay. This effect was also seen forextracts of Riso 1508 where the total stainable material running asprotein bands was diminished compared to Riso 56 or Bomi. Riso 1508 hasbeen shown to accumulate more non-protein N as free amino-acids (Koieand Kreis, 1978).

The cross-section of F2 seeds was also examined. When compared towild-type, in some cases the endosperm of the apparent double null seedsappeared moderately shrunken, in others more severely shrunken.

The second half of each of the F2 seeds were germinated on moist filterpaper, the F2 plantlets transferred to soil in the greenhouse and grownto maturity to provide F3 seed. Various plant growth and yieldparameters were measured (Table 2).

TABLE 2 Growth and yield parameters for F2 barley plants, rankedaccording to 100 seed weight for the F3 seed. B and C hordein No. ofHarvest 100 seed Weight Plant phenotype Height Tillers index Seeds/Head(% of K8) Sloop WT 36.34 ± 2.2   9.2 ± 0.97 0.60 ± 0.02 10.3 ± 0.7  5.47± 0.16 K8 WT  54.7 ± 1.16 34 0.63 ± 0.02 21.9 ± 1.4  4.65 ± 0.11 (100%)L1 WT  46.6 ± 0.94 40 0.66 ± 0.01 22.0 ± 1.1  4.41 ± 0.05 (94.8%) 9RE bcreduced  56.8 ± 2.14 11 0.56 ± 0.01 19.0 ± 1.24 4.19 ± 0.13 (90.1%)R1508 c null, 36.41 ± 0.34 27.5 ± 3.5 0.66 ± 0.01 20.0 ± 0.7  4.02 ±0.02 (86.5%) Red. B 5RB bc reduced  62.0 ± 2.24 28 0.46 ± 0.02 15.8 ±1.2  4.01 ± 0.01(86.2%) G1 bc reduced  61.4 ± 1.19 34 0.45 ± 0.02 16.0 ±1.0  3.83 ± 0.09 (82.4%) 5BD Red. B  63.9 ± 1.68 19 0.45 ± 0.01  15 ±0.9 3.70 ± 0.09 (79.6%) R56 b null 56.24 ± 0.34 20.0 ± 2.0 0.51 ± 0.0116.8 ± 1.2  3.70 ± 0.08 (79.6%) B5 WT  47.3 ± 1.36 34 0.52 ± 0.02 14.3 ±0.6  3.52 ± 0.12 (75.7%) J1 bc reduced  50.7 ± 1.71 32 0.57 ± 0.02 23.9± 0.4  3.56 ± 0.03 (76.6%) 4BH Red. B  44.9 ± 0.79 19 0.56 ± 0.01 19.7 ±0.6  3.29 ± 0.17 (70.7%) D6 bc reduced  42.3 ± 1.23 24 0.47 ± 0.02 9.2 ±0.8 2.90 (62.4%) 6RF Red. B  61.4 ± 1.66 23 0.35 ± 0.05 6.6 ± 1.6 2.86(61.5%) B1 WT  51.9 ± 2.79 12 0.37 ± 0.03 9.6 ± 1.5 2.62 ± 0.11(56.3%)J4 bc reduced 49.8 ± 0.59  17 0.35 ± 0.03 7.4 ± 1.1 2.64 ± 0.01 (56.8%)Red = reduced for specified hordein.

Plant height, head and stem weight, number of tillers, seeds per head,and 100 seed weight were measured. Harvest index was calculated from theratio of the head weight/(stem weight+head weight). The F3 seed werethen grown in the field to provide F4 seed of each line.

The F3 seeds showed a considerable variation in all measured parameterswhen compared to the parents and the control line, Sloop. Many of theapparent double null lines, such as J4 and 6RF, had 100 seed weightsreduced by up to about 40% or reduced numbers of seeds per head relativeto the wild type sibling K8. This suggested that there were other genessegregating in the population as well as the hordein B or C mutationshaving an effect on yield. However, several F3 lines had seed weightsgreater than or equal to the parents and therefore it was likely thatthe other genes could be segregated away from the B hordein and lys3amutations.

In cross section, the appearance of F3 seeds varied from shrunken(similar to Riso 1508) to slightly shrunken (similar to Riso 56) whencompared to wild type siblings or the control Sloop.

The total water soluble and alcohol-soluble proteins from eight F3 seedsfrom several lines were extracted as described above. The proteincontent of the alcohol soluble and aqueous soluble fractions wasmeasured as described in Example 1 using known amounts of gamma-globulinas a protein standard. However the total alcohol soluble protein levelsin some samples of F3 seeds were essentially the same as Riso 1508.Subsequently it was determined that these seed samples were segregatingfor the wild-type allele of the Lys3a gene and were not uniformly“double null”.

Quantitation of Hordein Levels in F3 Seeds by RP-FPLC

Alcohol soluble extracts from two seeds from each line were combined and50 μl examined by RP-FPLC as described above. The chromatograms areshown in FIG. 11. The total area under the chromatograms correspondingto hordein was calculated and expressed relative to levels in a wildtype line. The data (Table 3) showed that the F3 grain had hordeinlevels that were less than 30% of the wild-type level, in some casesless than 20%, even as low as 5.3%. The lack of substantial proteinbands following SDS-PAGE supports the contention that the total alcoholprotein levels were inflated due to elevated non-protein nitrogen levelsin the F3 seeds.

TABLE 3 Relative hordein levels in F3 seeds measured by RP-FPLC. LineHordein content Wild type (K8) 100%  R56 70% R1508 50% 4BH 26% 5RB 21%9RE 16% J1  5%

Example 4 Properties of Field-Grown F4 Barley Grain

The characteristics of glasshouse grown and field grown F4 seeds ofselected lines (9RE, J1, G1, 4BH), single null parents (Riso 56 and Riso1508), and wild type barley (Sloop; Bomi; and K8, a reconstituted wildtype sibling from the same cross as the double null lines) werecompared.

Seed Weight

The 100 seed weight of F4 seeds grown in the glasshouse varied from60-76% of Sloop (5.47+0.16 g per 100 seed), whereas the 100 seed weightof F4, field grown grain was lower, varying between 58-65% of Sloop(4.75 g+0.04).

Germination of Grain

Germination of seeds from two selected F4 barley lines was compared towild-type cv. Sloop by imbibing samples of 100 grain each on moist paperfor six days. Germination was observed as emergence of the root tip fromthe seedcoat. The F4 grains appeared to germinate at the same rate asthe wild-type grain, with about 60-70% germination after 3 days. Storageof the grain at 37° C. for 4 weeks prior to imbibition slightlyincreased the % germination of both F4 lines. Treatment at 4° C. for 3days also achieved the same increase over freshly harvested material.

This demonstrated that the grain of the F4 lines did not suffer anyserious retardation of germination, and therefore were predicted to beagronomically useful.

Protein Levels in F4 Grain

The levels of water-, salt-, alcohol-, and urea-soluble proteins ingrain of the F4 lines were measured using duplicate 20 mg samples ofwholemeal flour from glasshouse grown, F4 seeds of selected lines (9RE,J1, G1, 4BH), single null parents (Riso 56 and Riso 1508), and wild typebarley (Sloop; Bond; and K8).

Water-soluble proteins were extracted from each flour sample using 0.5nil of water, by mixing for 30 min, centrifuging the mixture at 13,000rpm for 5 minutes, removing the supernatant, and repeating theextraction on the pellet twice. The supernatants were pooled(water-soluble extract) and the pellet sequentially extracted threetimes in the same manner using 0.5 ml of 0.5M NaCl (salt-solubleextract), followed by 0.5 ml of 50% (v/v) propan-1-ol containing 1%(w/v) DTT (alcohol-soluble extract (hordeins)), followed by 8M ureacontaining 1% (w/v) DTT (urea-soluble extract). The protein content ofeach fraction was measured by using a dye binding assay (BioRad)according to the manufacturer's instructions, calibrated against gammaglobulin as a protein standard. The data are shown in FIG. 12. The totalextractable protein content (FIG. 12E), was calculated from the sum ofthe protein contents of all the soluble fractions.

In addition the total nitrogen (Total N; FIG. 12F) was measured usingduplicate 2.5 mg samples of the same flour by elemental analysisfollowing combustion at 1800° C. and reduction to N₂ at 600° C., andquantification by mass spectroscopy (method of Dumas). The total proteincontent was calculated using the expression: protein content=6.63×amountof total N. The figures obtained for total protein levels by MS werereasonably similar to the estimated total extractable protein contents,showing that the protein extraction was efficient.

The hordein content (measured as the level of alcohol-soluble protein)of the F4 grain was reduced to 17-39% of the parents (R1508 and R56) andto 7-16% of the wild type cultivar Sloop. This represented about a10-fold reduction in the level of total hordeins, shown above to betoxic to coeliacs, in these grain samples relative to wild type barley,Sloop.

The other types of proteins, in particular the water- and salt-solubleproteins are thought to have beneficial effects on the brewingproperties of barley grain. Since the levels of water- and salt-solubleproteins of the F4 grains were similar to those in the wild-type, Sloop,it was considered that the F4 grains would have sufficient of theseproteins for brewing purposes.

Fatty Acid Content and Composition

Since a major nitrogen sink during growth and development of the seedshad been removed by reducing the hordeins, the mutant grain was analysedto determine whether the developing seed might have compensated byincreasing the storage of for other components, some of which could bedeleterious to use of the grain. The fatty acids in duplicate 50 mgsamples of wholemeal flour from F4 grain were extracted, methylated andanalysed by quantitative gas chromatography (GC) using the method ofFolich et al. (1957).

The total fatty acid concentration in the F4 grain of lines G1, BB5, J1,and J4 varied in the range from about 2.5% to 3% (w/w), and was similarin level to that in the single null and the wild type barley grain, Itwas concluded that the double null grains did not contain elevatedlevels of fatty acids.

The fatty acids in the grain lipid comprised mainly linoleic (C18:2),oleic (C18:1) and palmitic acids (C16:0), with lower levels of otherfatty acids. There was no significant difference in the concentrationsof individual fatty acids that had accumulated in the selected F4 graincompared to the single null parents or the wild type barley. Inparticular, the concentration of erucic acid (C22:ln-9), which is toxicto humans in high concentration, in the F4 grain was not increased. Themutant grain therefore had normal fatty acid content and composition.

Starch Levels

Starch is the main component of cereal grain, typically comprising about55-65% of the dry weight. Starch levels are particularly important inbarley which is used for malting. A starch content which is too low mayresult in the formation of malt which has insufficient sugar content toenable efficient fermentation to occur during brewing, and thereforestarch levels in the barley grain were measured.

Starch of the mutant grain was isolated and assayed essentially asdescribed in the Megazyme Method (AACC76.13) using 20 mg of whole mealflour samples. Total starch levels in the F4 grain were in the rangefrom 57% to 66% (w/w), and were similar to the starch content of thesingle null parents and the wild type barley which were in the range of51-64% (w/w).

It was concluded that the F4 barley grains had sufficient starch toenable production of malt from the grain.

β Glucan levels

The β-glucan content in the mutant grain was assayed as described inMegazyme Method (AACC32.23), using 20 mg samples of wholemeal flour.β-glucan levels in the grain of lines G1, BB5, J1, J4 were in the rangefrom 1.2 to 2.6% (w/w), and were similar to the β-glucan content of thesingle null parental grain and the wild type barley grain which were inthe range from 2.4-3.3% (w/w).

High β-glucan levels are involved in the formation of chilling haze inbeer during storage. It was concluded that the β-glucan content of theF4 grains was not elevated when compared to wild-type grains and thelevels were unlikely to interfere with the brewing performance of thesegrains.

Free Amino Acid Levels

Increased accumulation of free amino-acids could possibly be deleteriousfor use of the grain. For example, free asparagine in sufficient amountsmight form the toxic compound acrylamide if heated to high temperaturesin the presence of starch.

The content and composition of free amino-acids in the grain wasmeasured using replicate samples of 20 mg wholemeal flour fromglasshouse grown, F4 seeds. Samples were dissolved in 0.1N HCl and analiquot was taken and dried, and amino acids analysed using the WatersAccQTag chemistry by the Australian Proteome Analysis Facility (Sydney).

The most prevalent amino acids in the barley flours were proline,asparagine, glutamic and aspartic acid in decreasing order, in the rangeof about 1.5 mg/g flour down to 0.5 mg/g flour. The free proline contentin the selected F4 grain was in the range 0.6-1.5 mg/g, and was similarto the free proline content of the single null parents and the wild typebarley which were in the range 0.2-1.2 mg/g. Levels of all other freeamino-acids were correspondingly similar in F4 and control grains. Inparticular, the free asparagine content in the F4 grain was about 0.5mg/g for lines G1, BB5 and J1 and about 1.0 mg/g in line J4. In thesingle null parental grains, the free asparagine level was 0.3 or 0.9mg/g, and in the wild type barley grains, free asparagine was in therange from 0.3-0.6 mg/g.

Since the free asparagine content of the F4 grain was similar to levelsin the corresponding wild type grain, it was considered that theproduction of acrylamide from free asparagine during malting or otheruse of the grain would be no different than for the wild-type grain.

Free lysine is known to be a limiting amino-acid in animal nutrition andtherefore levels of this amino acid were of interest for potential useof the grain as animal feed. The free lysine content in the F4 grain oflines G1, BB5 and J1 was about 0.5 mg/g and 1.0 mg/g in grain of theline J4. This represented a 181%-1,020% increase compared to the levelin wild-type grain of cultivar Sloop. Thus the F4 lines were a morenutritious source of free lysine than Sloop.

Example 5 Testing of F4 Grains—T-cell Toxicity Testing

To test the coeliac toxicity of the F4 grain, hordeins were isolated andpurified from 10 g samples of wholemeal flour from field grown seeds ofselected lines 9RE, J1, G1 and 4BH, single null parents (Riso 56 andRiso 1508), and wild type barley (Sloop; Bomi; and K8) as describedbelow. The purified hordeins were adding to T-cells isolated from apopulation of coeliacs to test for coeliac toxicity. The test involvedmeasuring the number of T-cells which produced gamma-interferonfollowing overnight incubation with the purified protein, using anantibody assay for the level of gamma-interferon. That is, the level ofgamma-interferon was an indication of the extent of toxicity of theproteins in the grain. This measure of the coeliac toxicity of the flourwas then plotted as a function of the fresh weight of flour obtainedfrom the grain.

Purification of Prolamins (Hordeins)

Wholemeal flour (10 g) was stirred for 30 min at 25° C. in 200 ml ofbuffer containing 20 mM triethanolamine-HCl (TEA), 1% (w/v) sodiumascorbate, 1% (w/v) polyethylene glycol (MW 6000; PEG 6000), and 1 μg/mlof protease inhibitors E64 and AEBSF (Sigma); the buffer adjusted to pH8. The suspension was centrifuged at 5,000 g for 5 min, the supernatantdiscarded and the pellet washed twice more. Proteins in the washedpellet were dissolved in 80 ml of 50% (v/v) propan-2-ol, containing 1%(w/v) DTT, by stirring for 30 min at 60° C. The suspension was chilledat 4° C. for 10 minutes and centrifuged at 10,000 g for 10 min at 4° C.The proteins including hordeins in the supernatant were precipitatedwith 2 volumes of propan-2-ol overnight at −20° C., and sedimented at10,000 g for 10 min at 4° C., and the pellet dissolved in 10 ml ofbuffer which contained 8M freshly deionised urea, 1% DTT, 20 mM TEA,adjusted to pH 6.

The hordeins were purified by FPLC as follows. The hordein solution (1ml) was injected into an 8 ml column of Source 15 Reverse PhaseChromatography (RPC, Pharmacia). The column was washed with 4 ml of 5%solvent B, and hordeins eluted with a 2.5 ml linear gradient from 5%solvent B to 35% solvent B at 4 ml/min, followed by a linear gradientfrom 35% solvent B to 83% solvent B over 36 mi. Solvent A was 0.1% (v/v)trifluoroacetic acid (TFA) in water, solvent B was 0.1% (v/v) TFA in 60%(v/v) aqueous acetonitrile. Fractions eluting between 25 and 43 nil werepooled. Solvent controls were similarly pooled from runs without aninjection. Corresponding pools from 10 sequential injections werecombined, and lyophylised.

Ex Vivo T-cell Assays

FPLC purified hordeins (50 mg/ml in 2M urea) were diluted with PBScontaining 1 mM CaCl₂, to give either 25, 62.5, 125, 250, 625, 3,750, or6250 μg hordein/ml and deamidated by adding 25 μl of each solution to100 μl of guinea pig liver tTG (Sigma; 25 μg/ml tTG in PBS containing 1mM CaCl₂) and incubated for 6 hr at 37° C. Non-deamidated solutions weresimilarly prepared by incubation in the absence of tTG. Solvent controlswere added as for the highest hordein concentrations. Other controlsamples contained either the solvent control, the solvent controlcontaining a known toxin, the tetanus toxoid (50 light forming units/ml,obtained from Commonwealth Serum Laboratories, Melbourne); or tetanustoxoid (50 light forming units/ml) alone. All solutions were then frozenat −20° C.

T-cells were obtained as follows. Six, biopsy-proven, HLA-DQ2⁺ coeliacsubjects, adhering to a strict gluten-free diet for at least threemonths, consumed 150 g of boiled barley daily for 3 days. PBMC wereisolated by Ficoll-Hypaque density centrifugation from heparinisedvenous blood collected either immediately prior to or six days followingcommencement of dietary challenge, and resuspended in complete HT-RPMIcontaining 10% heat-inactivated, pooled, human AB serum. Deamidated ornon-deamidated hordeins and control solutions were thawed and 25 μladded to wells containing 100 μl of PBMC (3-8×105 PBMC per well),cultured at 37° C. overnight in 96-well plates (MAIP-S-45; Millipore,Bedford, Mass.) and compared to control cultures (no addition) to whichwas added 25 n1 of PBS containing 1 mM CaCl₂ alone. Final hordeinconcentrations were 0, 1, 2.5, 5, 10, 25, 150, or 250 μg/ml. The highestfinal urea concentration was 10 mM. IFN-γ was visualised using secondaryantibodies as in manufacturers notes (Mabtech, Stockholm, Sweden) aspreviously described by Anderson et al. (2005), and spot forming units(SFU) counted using an automated ELISPOT reader (AID AutoimmunDiagnostika GmbH; Germany). Results are presented as the mean spotforming units (SFU)±S.E vs the equivalent weight of flour which wouldcontain the calculated amount of hordein. The hordein content of eachflour sample was calculated in Example 5, allowing calculation of theweight of flour.

Data was analysed by GraphPAD Prism and the curves of best fitcalculated and shown with means+S.E. The r² values for the data weregreater than 0.83, indicating a good fit between observed data and thecurve of best fit (FIG. 13).

Results

T-cells isolated from a single coeliac subject prior to a dietarychallenge were less responsive to prolamins added at 25 μg/ml thanT-cells isolated from the same individual after a dietary challenge withbarley. The mean SFU±S.E. of 29.5±3.0, and 104±15.9 were observed forT-cells isolated before and after a dietary challenge. This indicatedthat coeliac specific T-cells were induced by the dietary challenge.

Using T-cells isolated six days after the dietary challenge, thepositive control, tetanus toxoid, gave a consistent response in theabsence and presence of tTG (mean SFU±S.E. 28.1±5.9 and 20.2±7.4,respectively). Addition of the solvent control did not significantlyinhibit the response of the positive tetanus toxoid control (meanSFU±S.E. 20.5+4.1 and 17.6±6.0 in the absence and presence of tTGrespectively) confirming that solvent impurities did not generate falsenegatives or inhibit the positive responses.

T-cells isolated from coeliacs, 6 days post challenge responded morestrongly to all tTG treated hordein fractions when compared to T-cellsexposed to non-deamidated hordeins as expected for coeliac disease(Hadjivassiliou et al., 2004, Kim et al., 2004) (FIG. 13A; for claritythe response to only two hordein samples, Sloop and G1, are shown). Thisconfirmed that the T-cell response being measured was related to coeliactoxicity.

As the concentration of hordein was increased, the number of SFU alsoincreased in a hyperbolic manner as expected for normal Michaelis-Mentenenzyme kinetics between an enzyme and its substrate. Two parameters aregenerally used to describe such curves: Bmax, the maximum number of SFUexpected at the highest concentration; and Kd, the concentration ofprotein required to induce half maximal SFU. The more toxic the floursample, then the lower the Kd.

The coefficients Kd and Bmax were calculated from the curves of bestfit. The Bmax values did not vary significantly between wild-type andmutants, as expected. In contrast, the Kd values for the F4 lines werehigher by a factor of 10 compared to the wild type lines (Table 4). Thatis, approximately 10 times more flour from the mutant lines was requiredto induce half maximal toxicity response than for the wild-type flour(Table 4). Thus it was concluded that the coeliac toxicity of the F4grain had been reduced by about 10-fold compared to the wild type lines.This level of reduction compared well with the decreased hordein levelfound by protein determination in the F4 grain.

TABLE 4 T-cell toxicity of barley flour. Kd Line (mg of flour for halfmaximal spots) Wild Type: Sloop 0.18 ± 0.03 Bomi 0.18 ± 0.02 Singlenull: Riso56 0.47 ± 0.09 Riso1508 3.31 ± 0.47 F4 lines: G1 2.3 ± 0.3 5RB2.6 ± 0.5 4BH 1.7 ± 0.2 J1 1.4 ± 0.2

The toxicity of the F4 grain was lower than that of Riso 56 as expected.However the toxicity of the F4 grain was similar to that of the otherparent Riso 1508. Subsequently, on further genetic characterisation ofthe F4 grain, it was found that this was due to heterozygosity of themutation of the gene encoding B-hordein protein in the selected F4lines, which had the effect of elevating the hordein content above thatexpected.

Example 6 Malting of F4 Grain

To determine the suitability of the barley grain for malting, analysesincluding small-scale malting (micro-malting) tests were carried out.

One factor that influences malting ability is seed size. Samples fromthe F4 grain were analysed for seed size distribution by counting theproportion of 1,000 seeds which were retained by 2.8, 2.5 or 2.2 mmscreens. The F4 grain on average were smaller than wild-type and similarto the parental grains Riso 1508 and Rico 56 with less than 5% of theseed retained by a 2.5 mm sieve (Table 5). This contrasted to thecontrol lines Galleon and Sloop where 90% of seed was greater than 2.5mm. It was noted that grains of K8 which is a wild-type line derivedfrom the same Riso 1508× Riso 56 cross were also reduced in size,therefore at least part of the reduction in seed size was related to thegenetic background and not directly due to the reduced hordein level. Inaddition, the smaller seed size could be compensated for bymodifications in the method for steeping of the grain.

TABLE 5 Size of seed used for micromalting. % seed population Lineretained by 2.5 mm sieve G1 1.0 4BH 2.6 5RB 3.2 J1 2.0 9RE 6.2 Riso 15084.0 Riso 56 6.9 K8 24.8 Bomi 56.6 Carlsberg II 57.5 Galleon 83.9 Sloop91.6

Seed moisture levels may affect the malting performance. The % moistureand % nitrogen were measured by Near-Infra-Red (NIR) analysis prior tomicro-malting. The level of seed moisture of all the F4 grain sampleswas in the range between 11 and 11.4% and was similar to the controllines except for grain of cv. Galleon (GA1, 8.9%). Seed nitrogen for thedouble null lines ranged between 2.3% and 2.5% which was higher than thecontrol malting line cv. Galleon, at 1.6%. For malting, seed nitrogenlevels is optimally between 1.5 and 2.0%.

Barley samples (170 g) from field grown, F4 grain from the selectedlines 5RB, G1, J1, 9RE, 4BH, single null parents (Riso 56 and Riso1508), and wild type barley K8, cultivars Bomi, Carlsberg II, Sloop andGalleon were steeped at 16° C. by soaking for 6 hrs, followed by restingfor 7 hr in air, followed by soaking for 6 hr, and then germinated at15° C. for 4 day in a JWM micromalting system. The germinated grain waskilned for 21 hr at a minimum temperature of 50° C., and a maximumtemperature of 80° C., and the resulting malts were cleaned of theirroots by rubbing and sieving.

The malts were analysed for moisture (%), total nitrogen (% dry wt) bywhole grain NIR and yield (expressed as weight of cleaned malt as apercentage of initial weight of barley).

In addition, malt samples were ground in a hammer mill and 50 g samplesdissolved in water heated from 45° C. to 70° C., to give 450 g finalweight of solution which was analysed for extract (% of grain weightsolubilised), colour, soluble nitrogen (N), Kohlbach index (KI: %soluble protein/total protein), β-glucan, viscosity, AAL (apparentattenuation limit or fermentability, % drop in density duringfermentation with brewers yeast), each according to the standardEuropean Brewery Convention protocols, ebc-nl.com (Table 6).

The protein content of the malts was generally higher than desirablespecification. This was shown by the total malt N, and the soluble N,however the proportion of soluble protein compared to the total (KI) wasclose to specification. The colour and viscosity of the F4 worts wasclose to specification and the β-glucan levels in the worts were low.These features were acceptable for malting.

The malting process involved three stages: malting, waitingfermentation. The overall efficiency is calculated from three measuresof the efficiency of each stage: yield, extract, and AAL respectively.These indicate that as each stage the F4 grain are approximately 10%less efficient than the benchmark grain, cv Galleon. Overallapproximately 1.3-fold more grain of the F4 lines would be required toproduce beer of a strength equivalent to the commercial standard,compared to Galleon.

All of these indications showed that malt could be made from the F4grains.

TABLE 6 Malt and wort analysis. Malt Analysis Wort Analysis (accordingto EBC) Moisture Total N Yield Soluble N βGlucan Viscosity AAL Line % %dry wt % Extract Colour % dry wt KI mg mPa.sec % Specification <5% 1.44-1.9 >85 >80%   3.0-4.5 0.6-0.8 38-46 <180 >1.6 >82 5RB 4.6 3.04 7569.3 12.4 1.57 52 25 1.54 75.0 G1 4.9 3.18 77 69.2 6.1 1.64 52 24 1.4771.8 J1 4.5 3.15 75 71.2 6.1 1.47 47 27 1.49 74.2 9RE 4.0 2.88 80 75.15.1 1.37 48 41 1.47 70.0 4BH 4.1 2.82 79 71.1 5.0 1.37 49 34 1.47 71.7Riso 1508 3.9 2.63 84 75.2 4.5 1.25 48 89 1.43 70.7 Riso 56 4.0 3.54 8372.1 3.8 1.11 31 194 1.40 75.9 K8 4.1 2.78 86 73.7 2.9 0.79 28 396 1.6467.9 Bomi 4.2 2.97 86 75.6 3.6 0.79 27 509 1.53 72.5 Carlsberg II 4.33.04 86 72.9 2.6 0.66 22 612 1.63 68.9 Galleon 3.8 1.59 88 81.5 3.3 0.5836 233 1.56 79.2 Sloop 4.0 2.73 86 74.2 3.2 0.87 32 229 1.56 77.6

Example 7 ELISA Analysis of Raw Malt Samples

Approximately 40 ml samples of wort from Example 6 were frozen,lyophylised, and dissolved in 20 ml of 6M urea, 1% (w/v) DTT, 20 mM TEA(pH 6) at room temperature. The protein content of each sample wasdetermined using the dye binding method of Bradford. Serial dilutionscontaining 20 μg of malt protein in 100 μl of 6 M urea, 1% DTT, and 20mM TEA (pH 6) were applied to a nitrocellulose membrane (Amersham HybondC+) which had been pre-equilibrated in PBS buffer, in a dot blotapparatus (BioRad) and calibrated with a purified C-hordein standard (2μg). The solution was drawn through the membrane under reduced pressure,and the membrane rinsed with PBS buffer containing 0.1% Tween 20 (PBST),the apparatus dissembled and the membrane blocked by incubating in 5%(w/v) skim milk powder in PBS buffer containing 0.1% Tween 20, for 1 hrat room temp. Hordeins were detected with a primary antibody (rabbitanti-wheat gliadin, antibody conjugated to horseradish peroxidase, fromSigma), diluted 1 part in 2000 of PBST buffer, for 30 min at roomtemperature. The membrane was washed with three changes of PBST bufferand developed by incubating in 10 ml of a 1:1 (v/v) mixture of reagentsAmersham ECL western blotting reagents A and B (GE HealthCare) and thesignal detected by exposing to Amersham Hyperfilm for 30 sec. The filmwas developed and quantitated using Total Lab TL100 software (Non-lineardynamics, 2006).

The raw malt solutions produced from the selected F4 grain had a meanlevel of hordein of 58±12.7 ppm.

This level was substantially lower than the limit of 200 ppm set byFSANZ for low gluten food in Australia and considerably lower than themean of 687±158 ppm found for malt from the wild type cultivars Galleon,Sloop, K8, Bomi, and Carlsberg II. It was also considerably lower thanthe hordein content of malt made from the parents Riso 56 and Riso 1508.

Ordinarily, the gluten (hordein) content of mixtures falls dramaticallythrough the malting, worting and fermentation processes, and finalstabilised beer may contain 1/1000 the level present in raw malt(Dostalek et al., 2006).

Therefore it was predicted that the hordein level in processed beer madefrom the F4 malts would be reduced to approximately 0.05 ppm, well belowthe range of 3-40 ppm found for beers made from wild-type barley grain(Dostalek et al., 2006).

There are several recent recommendations in the literature for the limitof gluten in the diet of coeliacs. The most reliable of these is basedon a multi-centre, placebo controlled, double blind trial and shows thatconsumption of less than 10 mg/day is safe for coeliacs; and recommendsthat consumption should be kept to less than 50 mg/day (Catassi et al.,2007). Another recent study confirms these findings and (Collin et al.,2004) advises that consumption of food with 100 ppm gluten would resultin consumption of about 30 mg/day and result in little damage tocoeliacs. FSANZ sets the food standards for New Zealand and Australia.The Codex Alimentarius Commission was created in 1963 by FAO and WHO todevelop food standards, guidelines and related texts such as codes ofpractice under the Joint FAO/WHO Food Standards Programme and is theaccepted statutory regulation for Europe, and North America. The Codexcurrently sets a gluten free limit of less than 0.05 g N (as gluten) per100 gm of food. There is a proposal to revise the Codex standard andproposes a limit of 20 ppm fcr food made from non gluten containingcereals, and 200 ppm for food made from gluten containing cereals (p32,PROPOSAL P264, REVIEW OF GLUTEN CLAIMS WITH SPECIFIC REFERENCE TO OATSAND MALT, FSANZ web site:foodstandards.gov.au/_srcfiles/P264_Gluten_Claims_FAR.pdf#search=%22gluten%20free%22).

It was concluded from the above analysis that consumption of beerproduced from the F4 barley lines would be well below the safety limitset for gluten free food for coeliacs, in the above studies andincluding the regulations set by FSANZ and the Codex Alimentarius.

Example 8 Further Characterisation of the F4 Lines

Alcohol soluble proteins were purified from bulk F4 seed harvested foreach of the indicated lines, as described above. Purified proteinsamples (20 μg) from the F4 grain of lines G1, J1, 4BH, 5RB and 9RE weredissolved in 6M urea, 2% (w/v) SDS, 1% (w/v) OTT, 0.01% (w/v)bromophenol blue, 0.0625 M Tris-HCL (pH 6.8) at 25° C., examined bySOS-PAGE, stained with 0.006% colloidal Commassie Blue, and compared tohordeins isolated from Riso 56, Riso 1508, and wild type lines (K8).Migration was compared to molecular weight standards to determinemolecular mass (Table 7).

Protein sequences were obtained by mass spectroscopy of tryptic digestsfrom protein spots cut from the gels, and processed for proteinsequencing by MS-MS fragmentation as previously described (Campbell etal., 2001) with a search against the NCBI non-redundant database.

TABLE 7 Protein identification from SDS-PAGE. Matched peptides SummedSpot (% NCBI MSMS no. ID ^(A) protein) Accession score Confidence^(E) 3D-hordein 15 (20%) 30421167 205 Certain 4 B3-hordein^(B)  9 (27%) 82371122 Certain 5 gamma-3-  3 (11%) 1708280 47 Reliable hordein^(C) 6garnma-hordein- 1 (2%) 123464 14 Indicative of 1 precursor homology only7 gamma-hordein-  6 (24%) 123464 94 Certain 1 precursor 8 gamma-3- 14(30%) 1708280 199 Certain hordein^(D) ^(A) All digests also containpeptides from porcine trypsin, as expected. ^(B)Also contained a lowlevel of D-hordein ^(C)Also contained low level of B-hordein ^(D)Alsocontained low level of gamma-hordein-1 precursor ^(E)The summed MSMSsearch score indicates the confidence of the identity assignment. Frompast experience, a score of over 15 is required for a reliableidentification, and a score of over 50 indicates almost certainidentification.

Peptides from each sample were bound to an Agilent Zorbax SB-C18 5 μm150×0.5 mm column with a flow rate of 0.1% (v/v) formic acid/5% (v/v)acetonitrile at 20 μl/min for one min then eluted with gradients ofincreasing acetonitrile concentration to 0.1% (v/v) formic acid/20%(v/v) acetonitrile over one min. at 5 μl/min, then to 0.1% (v/v) formicacid/50% (v/v) acetonitrile over 28 min, then to 0.1% (v/v) formicacid/95% (v/v) acetonitrile over one min. The column was washed with agradient from 0.1% (v/v) formic acid/95% (v/v) acetonitrile to 0.1%(v/v) formic acid/100% (v/v) acetonitrile over 5 min at 20 μl/min andre-equilibrated with 0.1% (v/v) formic acid/5% (v/v) acetonitrile for 7min before peptides from the sample were applied.

Eluate from the column was introduced to an Agilent XCT ion trap massspectrometer through the instrument's micronebuliser electrospray ionsource. As peptides were eluting from the column, the ion trap collectedfull spectrum positive ion scans (100-2200 m/z) followed by four MS/MSscans of ions observed in the full spectrum according to theinstrument's ‘SmartFrag’ and ‘Peptide Scan’ settings. Once twofragmentation spectra were collected for any particular m/z value it wasexcluded from selection for analysis for a further 30 sec to avoidcollecting redundant data.

Mass spectral data sets matched with sequence databases using Agilent'sSpectrum Mill software (Rev A.03.02.060). False positive matches wereavoided by using the software's ‘autovalidation’ default settings. Thisincludes a requirement for the peptide matches to be considerably betterthan the best match against the reversed database and various weightingsfavouring more probable ionisation and fragmentation patterns (‘protonmobility scoring’). Oxidised methionine was allowed as a variablemodification.

The results of protein sequencing established that the F4 seed from theselected lines unexpectedly contained a B3-hordein band, in addition togamma1-hordein and D-hordein as expected. The identity of the gamma-1and -3 hordein bands were established by sequencing proteins from theRiso 56 mutant where these proteins were not masked by co-migratingB-hordein bands. This indicated that the selected F4 lines were notcompletely lacking the B3 hordein.

Example 9 Identification of Barley Grain Lacking B and C Hordeins

Individual half seeds from a single head of field grown, F4 plants ofline G1 were swollen overnight in water containing protease inhibitorsE64 and AEBSF (1 μg/ml), individually crushed and ground in a plasticmicrotube with a stainless-steel ball, shaken at 30/sec for 3×1.5 min.in a 96 well Vibration Mill (Retsch Gmbh, Rheinische) and thencentrifuged at 3000 g for 5 min at RT and the supernatant discarded. Thewater-insoluble flour pellet was washed twice more in the same mannerand the supernatants discarded. Alcohol soluble hordeins in the pelletwere then extracted by adding 400 μl of 50% (v/v) aqueous propan-2-olcontaining 1% (w/v) DTT, followed by shaking and centrifugation asabove. Supernatants containing extracted hordeins were transferred tofresh tubes and the protein content in the DTT/propan-2-ol supernatantsmeasured with Coomassie reagent (BioRAD).

An aliquot of each hordein extract corresponding to 20 μg of hordein waslyophylised under vacuum overnight, dissolved in 15 μl of SDS-boilingbuffer, heated for 3 min at 90° C., loaded on a precast 12-18% Excellgradient gel (Pharmacia) and examined by SDS-PAGE as described inExample 1. A prominent band at approximately 43 kDa was observed tosegregate in individual seeds and was absent in extracts of 5 out of 16seeds. The position of this band was the same as the B3-hordein bandidentified previously.

The protein data confirmed that the F4 seed from line G1 washeterozygous and segregating for one or more B-hordein proteins. Thissituation was also confirmed for other F4 lines.

Genetic Testing

Genetic tests were carried out to confirm the protein data. Individualhalf-seeds from field grown, selected F4 lines were germinated in moistsoil, and grown for 2 weeks in the glasshouse at 25° C. days and 20° C.nights. DNA was isolated from 0.5 cm of the leaf blade using aREDExtract-N-Amp Plant PCR Kit (Sigma) according to the instructions.Gene sequences specific for B1-hordeins and gamma-hordeins wereamplified by separate PCR reactions by adding 10 μl of RMix, 1 μl eachof the B1-hordein primers (5′B1hor and 3′B1hor) or 0.5 μl each of thegamma3-hordein primers (5′gamma hor3 and 3′gamma 3-full), 4 μl plant DNAand MilliQ water to 20 μl, at room temperature and then subjected to thefollowing temperature programme in an Eppendorf thermal cycler: 95° C.for 10 min; followed by 35 cycles of 95° C. for 30 sec, 56° C. for 30sec, and 72° C. for 1 min. This was followed by 72° C. for 10 min, andcooling to 10° C.

The sequences of the PCR primers were as follows:

(SEQ ID NO: 2) 5′B1hor: 5′-CAACAATGAAGACCTTCCTC-3′ (SEQ ID NO: 3)3′B1hor: 5′-TCGCAGGATCCTGTACAACG-3′ (SEQ ID NO: 4) 5′ gamma hor3:5′-CGAGAAGGTACCATTACTCCAG-3′ (SEQ ID NO: 5) 3′ gamma 3-full:5′-AGTAACAATGAAGGTCCATCG-3′.

20 μl of each PCR reaction was loaded on a 1 cm, 1% (w/v) agarose gelcontaining EtBr, electrophoresed at 100 V for 1 hr in TBE buffer and animage obtained of the fluorescence of the DNA products using GelDocimage system (uvitec) (FIG. 14).

An amplified DNA band for the gamma3 hordein control gene was present inall lanes as expected (FIG. 14, lower panel, gamma3-Hor). AmplifiedB-hordein DNA was absent in all PCR lanes from Riso 56, as expected thegene has been deleted in Riso 56 (FIG. 14, top panel, R56). AmplifiedDNA bands for B-hordein genes segregated in extracts from seeds of asingle head of F4 lines 9RE and 4BH (FIG. 14, top panel 9RE, 4BH). Thisindicated that one or more B-hordein genes were present in some of theF4 seed and that the F3 seed were not homozygous for the deletion of theB-hordein locus in Riso 56. This was also shown for other F4 lines.

This method was useful as a DNA-based method to identify and selectseeds lacking the B1 hordein.

The results of the genetic testing were used to select for plants thatdid not contain B-hordein genes. Twelve individual F5 plants, null byPCR for B-hordein genes were selected, and grown to produce a populationof F5 seeds known as G1*. Individual G1*, F5 half seeds, were taken froma single head, germinated in moist soil and grown for 2 weeks in theglasshouse at 25° C. days and 20° C. nights before DNA isolation/PCRanalysis as above. The corresponding half-seed was used for hordeinisolation and analysis by taking an aliquot corresponding to 40 μg ofhordein, lyophylised under vacuum overnight, dissolved in 15 μl ofSDS-boiling buffer, heated for 3 min at 90° C., loaded on a precast 12%Longlife, 1 mm (Longlife Gels) and electrophoresed at 150V for 40 minand stained as in Example 1.

The PCR analysis showed that DNA isolated from the positive controllines, Sloop and Riso 1508 gave a B-hordein band as expected. The sizeof the band from Sloop was larger than that amplified from Riso 1508,since the B1-hordein genes were slightly different. The control gene,gamma3-hordein, was amplified from all plants. The PCR band was notamplified from extracts of six G1* individuals confirming the absence ofthe gene from these plants. The hordein pattern in the correspondinghalf seeds confirmed this; no B-hordein bands were observed in G1*.Therefore it was concluded that G1* lacked detectable B-hordeins and wasinferred to be a homozygous null for the locus encoding B-hordein.

The remaining 250 F5 G1* seeds were germinated and the seedlings testedand confirmed as null for the B-hordein gene. Subsequent generationswere used for seed increase of this line.

Analysis of Hordein Content

The barley varieties Sloop, R56, R1508 and G1* were grown in adjacentplots in the field, the mature grain harvested and processed to makeflour. Hordein levels in the flour samples were analysed as describedabove. Protein fractions soluble in water, salt solution, alcohol/DTTand urea soluble were obtained as in Example 4 and the protein contentin each measured. The protein contents are shown in Table 8, andexpressed as mg protein/gm dry weight flour. Each total protein contentwas determined by summing the protein content of the fractions for thatsample. Hordeins were contained in the alcohol soluble fraction alongwith other alcohol soluble proteins such as serpins, proteaseinhibitors, LTP1 and Protein Z.

TABLE 8 Protein content in fractions in flour obtained from G1* grainBarley Water Salt Alcohol/DTT Urea Variety soluble soluble (% Sloop)soluble Total Sloop 17.2 17.6 23.1 (100%) 48.0 106 R56 16.7 19.0 13.2(58%)  58.6 108 R1508 22.2 15.0 8.0 (35%) 53.5 99 G1* 19.0 22.2 4.8(21%) 58.6 105

The data showed that the alcohol soluble protein content in G1* grainand consequently the flour was reduced to less than 22% relative to thewild-type cultivar Sloop.

The alcohol soluble protein fractions obtained above were enriched forhordeins by FPLC as in Example 5. The proteins in each FPLC eluate werelyophylised and the yield of FPLC-purified protein per 10 g of flourdetermined. This showed that the hordein content of G1* was reduced toless than 8 mg/10 g flour compared to 105 mg/10 g flour for Sloop, 38for R56 and 24 for R1508. This represented a reduction in the hordeincontent in G1* grain and flour of at least 92% relative to Sloop.

Example 10 Larger Scale Malting and Brewing Using F4 Grain

Larger scale malting experiments were carried out to produce sufficientquantities of malt for brewing tests using the F4 grain. These testsused modified steeping procedures, to take account of the smaller grainsize amongst other factors, as follows. Grain samples of 800 g permalting tin were used. Steeping regime was 17° C. for 5 hours,germination temperature was 15° C. for 94 hours. The kiln program was50-78° C. for 17 hours, 50-74° C. for 17 hours. Malt production did notuse gibberellic acid, this was not needed.

Mashing recipe: 4.65 kg low gluten malt, 10 litres water, 10 g calciumchloride, 2 g calcium sulphate, 64-65° C. for 2 hours.

Kettle: add 17 g Target Hop Pellets (10.0% AA) for 60 min, 21 gHallertau Hop Pellets (4.5% AA) for 10 minutes.

Fermentation was in 19 litre batch volume, at 12° C. fermentationtemperature, using 12 g Fermentis W34/70 dry yeast, for 8 days primaryfermentation, then 9 days chilled at 0° C. The beer was then filteredthrough a 1 micron filter, force carbonated in a keg, and filled with acounter-pressure bottle filler. The original specific gravity was 1.044,final gravity of fermented product was 1.013 with an ApproximateBitterness of 30 IBU, and the Approximate Alcohol by volume was 4.0%.

Other parameters measured during the production process were as follows:Malt moisture: 4.2%, Extract 71.5; Colour 3.9; WC 1.0; TN 2.63% drybasis; SN 1.11; KI 51; Viscosity 1.52; AAL 71.8%; beta-glucosidase 130mg/1; DP 24.

All of these indications showed that beer could be made from the maltfrom the F4 grains.

Larger scale malting and brewing tests were also performed on G1* barleygrain. Eight hundred gm of grain was malted in a Joe White Maltingsautomated malter, according to the indicated protocol. Preferred maltingconditions for G1* grain was determined to be: 3 hrs steeping at 17° C.,4 days germination at 15° C., followed by drying in a 50-80° C. kiln.The optimal length of time for steeping G1* grain differed slightlycompared to other grains: Sloop: 8 hr-9 hr-5 hr steep/rest/stepprogramme at 17° C.; R1508: 7 hr-8 hr-3 hr steep/rest/step programme at17° C.; R56: 8 hr-10 hr-5 hr steep/rest/step programme at 17° C.Analysis protocols were as specified by European Brewing Convention(EBC) or Institute of Brewing (IOB). Moisture content of the grain wasdetermined by Near Infrared spectroscopy (NIR). Total nitrogen contentwas determined by the method of Dumas. Data for the malts are shown inTable 9. One significant difference between the G1* grain and the othervarieties tested was that the diastatic power measures for G1* and R1508were much lower than for Sloop or R56 grain. This was thereforeassociated with the lys3 mutation in G1* and R1508.

Maltings were repeated and combined for each variety. Approximately 4 kgof malt from each of the lines G1*, R56, R1508 and Sloop (wild-type) wasbrewed and bottled as follows. The malt samples were bittered withTettnang hops for 60 min at boiling temperature to achieve 21-22 bitterunits (IBU). Fermentation was with US-05 yeast (Fermentis) at 18-20° C.The fermented product was kegged without filtration and force-carbonatedbefore bottling. All of the beers were still cloudy when bottled butwere clearer after 2-4 weeks storage. The beers had a noticeable“butterscotch” aroma and flavour due to diacetyl when kegged and bottledbut this also faded on storage.

Data for the brewed products are shown in Table 10. The alcohol contentfor the beer made from the G1* grain was 4.2% by volume. The G1* beerhad a slightly reduced, but satisfactory, head of foam after pouring.

TABLE 9 Data for malting characteristics of G1* grain. Peak % grain %Apparent β- moisture Malt Extract Wort Total Soluble KohlbachAttenuation Glucan Diastatic achieved Moisture EBC Colour Claritynotrogen nitrogen Index Viscosity Limit EBC power Variety % % fine EBCEBC (% d wt) EBC EBC EBC EBC mg/L IOB G1* 46.5 3.9 75.3 5.8 1.5 2.100.96 46 1.52 65.3 86 9 Sloop 45.7 4.1 80.7 3.1 1 1.94 0.86 44 1.55 76.9237 79 R1508 50.7 4.1 77.6 5.4 1 1.86 0.99 53 1.68 72.3 124 13 R56 48.13.9 79.6 4.3 1 1.77 0.78 44 1.53 78.5 240 63

TABLE 10 Data for characteristics of beer brewed from G1* grain. SLOOPR1508 R56 G1* Batch Volume (lt) 15.0 14.1 18.6 18.0 Malt Weight (kg)3.60 3.33 4.00 4.55 Protein Rest 57 C./20 56 C./20 54 C./20 55 C./20(Temp/Time) min min min min Amylase Rest 65 C./1 hr 63-65 C./1 64-65C./2 64-65 C./2 (Temp/Time) hr hrs hrs Original Gravity (SG) 1.051 1.0521.051 1.049 Final Gravity (SG) 1.014 1.013 1.012 1.017 Alcohol contentby 4.8% 5.1% 5.2% 4.2% volume (%) These experiments indicated that G1*grain could be successfully malted and brewed. Hordein levels in thebeer made from G1* grain, measured by immunoassay, are expected to beless than 1 ppm, and in some case less than 0.5 ppm. This compares to arange in hordein levels in wheat beer of 10-41 ppm, in stout of 9-15ppm, in lagers of 3-9 ppm.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 60/964,672, the entirecontents of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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The invention claimed is:
 1. A process of making a malt-based beverage,the process comprising obtaining malt from barley grain which ishomozygous for at least one genetic modification which results in 25% orless of the level of total hordeins when compared to a correspondingwild-tyke barley grain, mixing the malt with at least one other beverageingredient to make a mixture, and processing the mixture to produce themalt-based beverage, wherein the malt produced from said grain comprisesabout 25% or less of the level of total hordeins when compared to maltproduced in the same manner from corresponding wild-type barley grain.2. The process of claim 1, wherein the malt is processed so that it ismilled, stiffed into water and heated to produce a wort.
 3. The processof claim 1, further comprising brewing the malt and the at least oneother beverage ingredient after the malt and the at least one otherbeverage ingredient are mixed.
 4. The process of claim 1, wherein thebarley grain i) comprises about 15% or of the level of total hordeinswhen compared to grain of the corresponding wild-type barley plant, ii)has an average weight (100 grain weight) of at least 2.4g, iii) has anaverage weight (100 grain weight) of about 2.4g to about 6g, iv) has astarch content which is at least 50% (w/w), or v) has a starch contentwhich is about 50% to about 70% (w/w).
 5. The process of claim 1,wherein at least 50% of the barley grain germinates within 3 daysfollowing imbibition.
 6. The process of claim 1, wherein the at leastone ether beverage ingredient comprises sugar.
 7. The process of claim1, wherein the at least one other beverage ingredient comprises anunmalted cereal adjunct.
 8. The process of claim 1, wherein themalt-based beverage is beer.
 9. The process of claim 5, wherein the beercomprises at least 2% ethanol.
 10. The process of claim 1, wherein themalt-based beverage is whiskey.
 11. The process of claim 1, wherein themalt comprises about 15% or less of the level of total hordeins whencompared to malt produced in the same manner from grain from acorresponding wild-type barley plant.
 12. The process of claim 11,wherein the at least one other beverage ingredient comprises sugar. 13.The process of claim 11, wherein the at least one other beverageingredient comprises an unmalted cereal adjunct.
 14. The process ofclaim 11, wherein the malt-based beverage is beer.
 15. The process ofclaim 14, wherein the beer comprises at least 2% ethanol.
 16. Theprocess of claim 11, wherein the malt-based beverage is whiskey.
 17. Theprocess of claim 1, wherein obtaining the malt from barley graincomprises producing the malt from the barley grain by a processcomprising i) steeping the barley grain to produce steeped barley grain;ii) germinating the steeped barley grain to produce germinated barleygrain; and iii) drying the germinated barley grain.